Methods of treating viral infections using antiviral liponucleotides

ABSTRACT

Compounds are disclosed for treating AIDS, herpes, and other viral infections by means of lipid derivatives of antiviral agents. The compounds consist of nucleoside analogues having antiviral activity which are linked, commonly through a phosphate group at the 5′ position of the pentose residue, to one of a selected group of lipids. The lipophilic nature of these compounds provide advantages over the use of the nucleoside analogue alone. It also makes it possible to incorporate them into the lamellar structure of liposomes, either alone or combined with similar molecules. In the form of liposomes, these antiviral agents are preferentially taken up by macrophages and monocytes, cells which have been found to harbor the target HIV virus. Additional site specificity may be incorporated into the liposomes with the addition of ligands, such as monoclonal antibodies or other peptides or proteins which bind to viral proteins. Effective nucleoside analogues are dideoxynucleosides, azidothymine (AZT), and acyclovir; lipid groups may be glycolipids, sphingolipids, phospholipids or fatty acids. The compounds persist, after intracellular hydrolysis, as phosphorylated or non-phosphorylated antiviral nucleosides. The compounds are effective in improving the efficacy of antiviral nucleoside analogues by prolonging the antiviral activity after the administration of the drug has ended, and in preventing retroviral replication in HIV infections which have become resistant to therapy with conventional forms of the antiretroviral agents.

RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 08/483,148, filedJun. 7, 1995, now abandoned, which is a divisional of U.S. Ser. No.08/415,769, filed Apr. 3, 1995, now U.S. Pat. No. 6,448,392, which is acontinuation of U.S. Ser. No. 08/040,499, filed Mar. 31, 1993, nowabandoned, which is a continuation of U.S. Ser. No. 07/373,088, filedJun. 28, 1989, now U.S. Pat. No. 5,223,263, which is acontinuation-in-part of U.S. Ser. No. 07/319,485, filed Mar. 6, 1989,now abandoned, which is a continuation-in-part of U.S. Ser. No.07/216,412, filed Jul. 7, 1988, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates generally to the treatment of viralinfections using lipid derivatives of antiviral nucleoside analogues.More particularly, the present invention relates to lipid, andespecially phospholipid, derivatives of modified antiviral nucleosideanalogues which can be integrated into the structure of liposomes,thereby forming a more stable liposomal complex which can delivergreater amounts of drugs to target cells with less toxicity.

The publications and other reference materials referred to herein arehereby incorporated by reference, and are listed for convenience in thebibliography appended at the end of this specification.

There has been a great deal of interest in recent years in the use ofnucleoside analogues to treat viral infections. A nucleoside consists ofa pyrimidine or purine base which is linked to ribose, a five-carbonsugar having a cyclic structure. The antiviral nucleoside analoguesclosely resemble natural nucleosides and are designed to inhibit viralfunctions by preventing the synthesis of new DNA or RNA. Nucleosides areenzymatically assembled into DNA or RNA.

During DNA synthesis, free nucleoside triphosphates (nucleosides withthree phosphate groups attached) react with the end of a growing DNAchain. The reaction involves the linking of the phosphate group at the5′ position on the incoming nucleoside triphosphate with the hydroxylgroup at the 3′ position of the sugar ring on the end of the forming DNAchain. The other two phosphate groups are freed during the reaction,thereby resulting in the addition of a nucleotide to the DNA chain.

Nucleoside analogues are compounds which mimic the naturally occurringnucleosides sufficiently so that they are able to participate in viralDNA synthesis. However, the antiviral nucleoside analogues havestrategically located differences in chemical structure which inhibitviral enzymes such as reverse transcriptase or which prevent further DNAsynthesis once the analogue has been attached to the growing DNA chain.

Dideoxynucleosides are antiviral compounds that lack the hydroxyl groupsnormally present at the second and third position of ribose. When adideoxynucleoside is incorporated into a growing DNA chain, the absenceof the 3-OH group on its ribose group makes it impossible to attachanother nucleotide and the chain is terminated. Dideoxynucleosides areparticularly useful in treating retroviral infections where viralreplication requires the transcription of viral RNA into DNA by viralreverse transcriptase. Other nucleoside analogues includedeoxynucleosides and nucleosides analogues having only a fragment ofribose or other pentose connected to the base molecule.

Acquired immunodeficiency syndrome (AIDS) is caused by the humanimmunodeficiency virus (HIV). HIV infects cells bearing the CD4 (T4)surface antigen, such as CD4+ helper lymphocytes, CD4+ monocytes andmacrophages and certain other CD4+ cell types. The HIV infection of CD4+lymphocytes results in cytolysis and cell death which contributes to theimmunodeficiency of AIDS; however, CD4+ monocytes and macrophages maynot be greatly harmed by the virus. Viral replication in these cellsappears to be more prolonged and less cytotoxic than in lymphocytes, andas a result, monocytes and macrophages represent important reservoirs ofHIV infection. It has recently been discovered that macrophages mayserve as reservoirs of HIV infection even in certain AIDS patients whotest negative for the presence of HIV antibodies. No effective cure isavailable for AIDS, although dideoxynucleosides have been shown toprolong life and to reduce the incidence of certain fatal infectionsassociated with AIDS.

Certain monocyte-derived macrophages, when infected with some strains ofHIV, have been found to be resistant to treatment with dideoxycytidine,azidothymidine, and other dideoxynucleosides in vitro as shown byRichman, et al. (1). The resistance may be due in part to the low levelsof dideoxynucleoside kinase which result in a reduced ability tophosphorylate AZT, ddC or ddA. Clearly, it would be useful to have moreeffective ways of delivering large amounts of effective antiviralcompounds to macrophages infected with HIV or other viruses and othercells having viral infections. It would also be useful to have moreeffective ways of delivering antiviral compounds which not only increasetheir potency but prolong their efficacy.

Dideoxynucleoside analogues such as AZT are the most potent agentscurrently known for treating AIDS, but in a recent human trial, serioustoxicity was noted, evidenced by anemia (24%) and granulocytopenia (16%)(2,3). It is desirable, therefore, to provide a means for administeringAZT and other dideoxynucleosides in a manner such that the toxic sideeffects of these drugs are reduced. Further, it is desirable to provideselective targeting of the dideoxynucleoside to monocyte/macrophages toenhance the efficiency of the drug against viral infection in this groupof cells. One way to do this is to take advantage of the uptake ofliposomes by macrophages.

In 1965, Alex Bangham and coworkers discovered that dried films ofphosphatidylcholine spontaneously formed closed bimolecular leafletvesicles upon hydration (4). Eventually, these structures came to beknown as liposomes.

A number of uses for liposomes have been proposed in medicine. Some ofthese uses are as carriers to deliver therapeutic agents to targetorgans. The agents are encapsulated during the process of liposomeformation and released in vivo when liposomes fuse with the lipids ofcell surface membrane. Liposomes provide a means of delivering higherconcentrations of therapeutic agents to target organs. Further, sinceliposomal delivery focuses therapy at the site of liposome uptake, itreduces toxic side effects.

For example, liposomal antimonial drugs are several hundred-fold moreeffective than the free drug in treating leishmaniasis as shownindependently by Black and Watson (5) and Alving, et al. (6).Liposome-entrapped amphotericin B appears to be more effective than thefree drug in treating immunosuppressed patients with systemic fungaldisease (7). Other uses for liposome encapsulation include restrictionof doxorubicin toxicity (8) and diminution of aminoglycoside toxicity(9).

As previously mentioned, it is now thought that macrophages are animportant reservoir of HIV infection (10, 11). Macrophages are also aprimary site of liposome uptake (12, 13). Accordingly, it would bedesirable to utilize liposomes to enhance the effectiveness of antiviralnucleoside analogues in treating AIDS and other viral infections.

The use of liposomes to deliver phosphorylated dideoxynucleoside to AIDSinfected cells which have become resistant to therapy has been proposedin order to bypass the low dideoxynucleoside kinase levels.

Attempts have also been made to incorporate nucleoside analogues, suchas iododeoxyuridine (IUDR), acylovir (ACV) and ribavirin into liposomesfor treating diseases other than AIDS. However, these attempts have notbeen entirely satisfactory because these relatively small water solublenucleoside analogues tend to leak out of the liposome rapidly (14, 15),resulting in decreased targeting effectiveness. Other disadvantagesinclude the tendency to leak out of liposomes in the presence of serum,difficulties in liposome formulation and stability, low degree ofliposomal loading, and hydrolysis of liposomal dideoxynucleosidephosphates when exposed to acid hydrolases after cellular uptake of theliposomes.

Attempts have also been made to combine nucleoside analogues, such asarabinofuranosylcytosine (ara-C) and arabinofuranosyladenine (ara-A),with phospholipids in order to enhance their catabolic stability aschemotherapeutic agents in the treatment of various types of cancer(16). The resulting agents showed a decreased toxicity and increasedstability over the unincorporated nucleoside analogues. However, theresulting agents exhibited poor cellular uptake (16) and poor drugabsorption (17).

In order to use nucleoside analogues incorporated into liposomes fortreating viral infections more effectively, it is desirable to increasethe stability of the association between the liposome and the nucleosideanalogue.

In order to further enhance the effectiveness of these antiviralliposomes, it would be desirable to target the liposomes to infectedcells or sites of infection. Greater specificity in liposomal deliverymay be obtained by incorporating monoclonal antibodies or other ligandsinto the liposomes. Such ligands will target the liposomes to sites ofliposome uptake capable of binding the ligands. Two different approachesfor incorporating antibodies into liposomes to create immunoliposomeshave been described: that of Huang and coworkers (18) involving thesynthesis of palmitoyl antibody, and that of Leserman, et al. (19)involving the linkage of thiolated antibody to liposome-incorporatedphosphatidylethanolamine (PE).

The methods disclosed here apply not only to dideoxynucleosides used inthe treatment of AIDS and other retroviral diseases, but also to the useof antiviral nucleosides in the treatment of diseases caused by otherviruses, such as herpes simplex virus (HSV), human herpes virus 6,cytomegalovirus (CMV), hepatitis B virus, Epstein-Barr virus (EBV), andvaricella zoster virus (VZV). Thus, the term “nucleoside analogues” isused herein to refer to compounds that can inhibit viral replication atvarious steps, including inhibition of viral reverse transcriptase orwhich can be incorporated into viral DNA or RNA, where they exhibit achain-terminating function.

SUMMARY OF THE INVENTION

The invention provides a composition for use in treating viralinfections, including HIV (AIDS), herpes simplex virus (HSV), humanherpes virus 6, cytomegalovirus (CMV), hepatitis B virus, Epstein-Barrvirus (EBV), and varicella zoster virus (VZV). The composition maycontain, in addition to a pharmaceutically acceptable carrier, alipophilic antiviral agent prepared by chemically linking an antiviralnucleoside analogue to at least one lipid species. The antiviralnucleoside analogue may be linked to the lipid through a monophosphate,diphosphate or triphosphate group. The invention, further, provides amethod for incorporating such lipid derivatives of antiviral agents intoliposomes for improved delivery of the antiviral agent. A liposomecomprises a relatively spherical bilayer which is comprised wholly or inpart of the above-described lipid derivatives of antiviral agents. Theliposome may also contain pharmacologically inactive lipids. Further,the liposome may contain a ligand, such as a monoclonal antibody to aviral binding site (such as CD₄), or other binding protein. Such aligand provides additional specificity in the delivery site of theantiviral agent. The invention provides a method for incorporating suchligands into antiviral liposomes.

In one preferred embodiment, the compound is aphosphatidyldideoxynucleoside or a dideoxynucleoside diphosphatediglyceride. In another, the lipid species may comprise at least oneacyl ester, ether, or vinyl ether group of glycerol-phosphate.Phosphatidic acids having at least one acyl ester, ether, or vinyl ethergroup may also serve as a favored lipid species.

In another embodiment, the nucleoside analogue is a purine or pyrimidinelinked through a β-N-glycosyl bond to a pentose residue that lacks atleast one of the 2′ or 3′ carbons, but retains the 5′ carbon, and thephosphate group is bound to the 5′ carbon (i.e., what would have beenthe 5′ carbon in a complete pentose moiety). In another embodiment ofthe invention, the lipid species is an N-acyl sphingosine.

In some preferred embodiments, the acyl or alkyl groups of the lipidspecies, of whatever linkage, as for example ester, ether or vinylether, comprise 2 to 24 carbon atoms. In one variation, at least one ofthe acyl or alkyl groups is saturated. In another, at least one of theacyl or alkyl groups has up to six double bonds. In yet anotherembodiment, an acyl or alkyl group may be attached directly by ester oralkyl linkage to the 5′-hydroxyl of the nucleoside.

In still another, the lipid moiety is a glyceride and the glyceride hastwo acyl groups that are the same or different. In still anotherembodiment of the invention, the lipid species is a fatty alcoholresidue which is joined to a phosphate linking group through an esterbond. The compound may advantageously have from one to three phosphategroups, and at least one fatty alcohol ester, and may have two or morefatty alcohol residues that are the same or different in structure.These fatty alcohols are preferably linked to the terminal phosphategroup of the compound.

Moreover, the invention includes a composition wherein, in addition tothe compound, the liposome further comprises phospholipids selected fromthe group consisting of phosphatidylcholine, phosphatidylethanolamine,phosphatidylglycerol, phosphatidylserine, phosphatidylinositol andsphingomyelin.

In one embodiment of the invention, the percentage of antiviral agent is0.01 to 100 percent by weight of the liposome.

In another embodiment, the liposome further comprises a ligand bound toa lipid substrate. The ligand may be an antibody, such as a monoclonalantibody to a viral antigen. The viral antigen could be gp41 or gp110 ofHIV, or could be any other suitable viral antigen. In one embodiment,the ligand is CD4 receptor protein, or CD4 protein itself.Alternatively, the ligand is an antibody to CD4 or a protein or othersubstance that binds CD4.

The invention also contemplates a composition for use in treating viraland retroviral infections, comprising a liposome formed at least in partof an lipophilic antiviral agent, the agent comprising a nucleosideanalogue having a base and a pentose residue with at least one lipidspecies attached to the nucleoside analogue through a monophosphate,diphosphate or triphosphate linking group at the 5′ hydroxyl of thepentose residue of the nucleoside analogue, and a pharmaceuticallyacceptable carrier therefore.

Thus, there is provided a composition having antiviral properties,comprising an antiviral nucleoside analogue having a base portioncomprising a substituted or unsubstituted purine or pyrimidine, and asugar portion comprising a pentose residue, and a lipid moiety linked tothe pentose residue, with the proviso that the composition is in theform of a liposome when the pentose residue is ribose and the baseportion is cytosine, and when the pentose residue is arabinofuranose andthe base portion is cytosine or adenine. In one embodiment, thenucleoside analogue is a nitrogenous base which is a purine, pyrimidine,or a derivative thereof, and the pentose residue is a 2′,3′-dideoxy,2′,3′-didehydro, azido or halo derivative of ribose, or an acyclichydroxylated fragment of ribose. The pentose residue may thus be a2′,3′-dideoxyribose, and the nucleoside analogue may be2′,3′-dideoxycytidine, 2′,3′-dideoxythymidine, 2′,3′-dideoxyguanosine,2′,3′-dideoxyadenosine, 2′,3′-dideoxyinosine, or 2,6 diaminopurine,2′,3′-dideoxyriboside.

In another embodiment, the pentose residue is a 2′,3′-didehydroriboseand the nucleoside is 2′,3′-didehydrothymidine, 2′,3′-didehydrocytidinecarbocyclic, or 2′,3′-didehydroguanosine. In still another embodiment,the pentose residue is an azide derivative of ribose, and the nucleosideis 3′-azido-3′-deoxythymidine, 3′-azido-3′-deoxyguanosine, or2,6-diaminopurine-3-azido-2′,3′dideoxyriboside.

In still another embodiment of the invention, the pentose residue is ahalo derivative of ribose and the nucleoside is3′-fluoro-3′-deoxythymidine, 3′-fluoro-2′,3′-dideoxyguanosine,2′,3′-dideoxy-2′-fluoro-ara-adenosine, or2,6-diaminopurine-3′-fluoro-2′,3′-dideoxyriboside. The invention alsoincludes halo derivatives of the purine or pyrimidine rings, such as,for example, 2-chloro-deoxyadenosine. Alternatively, the pentose residueis an acyclic hydroxylated fragment of ribose, and the nucleoside is9-(4,-hydroxy-1′,2′-butadienyl) adenine, 3-(4,-hydroxy-1′,2′-butadienyl)cytosine, 9-(2-phosphonylmethoxyethyl)adenine orphosphonomethoxydiaminopurine.

In accordance with another aspect of the invention, the nucleosideanalogue is acyclovir, gancyclovir,1-(2′-deoxy-2′-fluoro-1-β-D-arabinofuranosyl)-5-iodocytosine (FIAC) or1(2′-deoxy-2′-fluoro-1-β-D-arabinofuranosyl)-5-iodouracil (FIAU).

In all of the foregoing compositions, a monophosphate, diphosphate, ortriphosphate linking group may be provided between the 5′ position ofthe pentose residue and the lipid species. Alternatively, there may bean aliphatic bridge comprising two functional groups and having from 0to 10 carbon atoms between the functional groups, the bridge joining thelipid and the pentose residue. In still further embodiments of theinvention, the lipid species is a fatty acid, a monoacylglycerol, adiacylglycerol, or a phospholipid. The phospholipid may have a headgroup comprising a sugar or a polyhydric alcohol. Specific examples ofphospholipids include bis(diacylglycero)-phosphate anddiphosphatidylglycerol. Other examples of lipid species includeD,L-2,3-diacyloxypropyl-(dimethyl)-beta-hydroxyethyl ammonium groups.

In accordance with another aspect of the present invention, the lipidspecies comprises from 1 to 4 fatty acid moieties, each the moietycomprising from 2 to 24 carbon atoms. Advantageously, at least one fattyacid moiety of the lipid species is unsaturated, and has from 1 to 6double bonds.

Particular examples of these compositions include3-phosphonomethoxyethyl-2,6-diaminopurine;1,2-diacylglycerophospho-5′-(2′,3′-dideoxy)thymidine.

Specific compositions are provided having the formula:

(L)_(m)—(W)_(n)—A—Q—Z

wherein

Z is the base portion of the nucleoside analogue, Q is the pentoseresidue, A is O, C, or S, W is phosphate, n=0 to 3, and L is a lipidmoiety wherein m=1 to 5, and wherein each L is linked directly to a Wexcept when n=0, in which case each L is linked directly to A.

Also included are compositions having the formula:

wherein Z is the substituted or unsubstituted purine or pyrimidine groupof the nucleoside analogue,

Q is the pentose residue,

W is phosphate, A is O, C, or S, L₁ is (CH₂—CHOH—CH₂), and

L is a lipid moiety.

In one embodiment of the invention, with reference to the foregoingformulas, each L is independently selected from the group consisting ofR,

wherein R, R₁ and R₂ are independently C₂ to C₂₄ aliphatic groups andwherein R, R₁ and R₂ independently have from 0 to 6 sites ofunsaturation, and have the structureCH₃—(CH₂)_(a)—(CH═CH—CH₂)_(b)—(CH₂)_(c)—Y

wherein the sum of a and c is from 1 to 23, and b is 0 to 6, and whereinY is C(O)O—, C—O—, C═C—O—, C(O)S—, C—S—, or C═C—S—.

In one embodiment of the foregoing compositions, the pentose residuecomprises ribose, dideoxyribose, didehydroribose, or an azido orhalosubstituted ribose, attached at the 9 position of the purine or atthe 1 position of the pyrimidine.

The present invention also provides a method for synthesizing a lipidderivative of an antiviral nucleoside, comprising the step of reactingan antiviral nucleoside, having a ribose hydroxyl group, with aphospholipid in the presence of a coupling reagent whereby thenucleoside is joined to the phospholipid by a phosphate bond at theposition of the ribose hydroxyl group. In one preferred embodiment, thephospholipid is a diacyl phosphate. In another, the phospholipid is aphosphatidic acid or a ceramide. Also provided herein is a method ofsynthesizing a lipid derivative of an antiviral nucleoside, comprisingthe steps of reacting an antiviral nucleoside monophosphate with areagent HL, wherein L represents a leaving group, to form a nucleosidePO₄-L, reacting the nucleoside PO₄-L with a phosphatidic acid to bindthe acid to the nucleoside through a pyrophosphate bond. In onevariation of the method, the nucleoside monophosphate is AZT5′-monophosphate.

Still a further method provided by the present invention is a method ofsynthesizing a glyceride derivative of a nucleoside analogue, comprisingthe step of joining a monoglyceride or diglyceride and an antiviralnucleoside monophosphate with a coupling agent in the presence of abasic catalyst. In one embodiment, the glyceride is 1-O-stearoylglyceroland the nucleoside is AZT monophosphate.

Also a part of the present invention is a method for preparing asuspension of liposomes for use in treating viral and retroviralinfections in a mammal, comprising providing a lipophilic antiviralagent comprising at least one lipid species attached to a nucleosideanalogue through a monophosphate, diphosphate or triphosphate linkinggroup at the 5′ position of the pentose residue of the nucleoside,combining the lipophilic antiviral agent and a pharmacologicallyacceptable aqueous solvent to form a mixture, and forming liposomes fromthe lipophilic antiviral agent. The liposomes may be formed, forexample, by sonication, extrusion or microfluidization. In one preferredembodiment, the combining step further comprises including in thecombination a pharmacologically inactive lipophilic lipid. This inactivelipid can be, for example, a phosphatidylethanolamine, a sphingolipid, asterol or a glycerophosphatide. The method also may include treating theliposomes with thio-antibodies to produce immunoliposomes, or includingin the combination an lipophilic lipid which is, in part, comprised of aligand. Thus, the liposome may include a ligand bound to a lipidsubstrate.

In addition, the invention includes a method for treating retroviral andviral infections in a mammal, such as a human, by administering asufficient quantity of the antiviral nucleoside analogues describedherein to deliver a therapeutic dose of the antiviral agent to themammal. In a preferred embodiment, the method is used to treatretroviral and viral infections in a mammal, wherein the retrovirus hasbecome resistant to therapy with conventional forms of an antiviralagent. The present invention also includes a method for treatment ofpatients having strains of HIV that have developed resistance to AZT orreduced sensitivity to AZT, comprising the step of administering acomposition of the present invention to such patient in an effective,retrovirus-inhibiting dosage. Also included in the present invention isa method for treating a viral infection in a mammal, comprising the stepof administering an effective amount of a composition as describedherein to a mammal. The infection may be a herpes simplex infection, andthe composition may be phosphatidylacyclovir. Alternatively, the virusmay be HIV retrovirus, and the composition may be 5′-palmitoylAZT. Themethod includes use where the retrovirus is a strain of HIV that hasdeveloped resistance to a nucleoside analogue.

Also disclosed herein is a method for prolonging the antiviral effect ofa nucleoside analogue in a mammal, comprising administering thenucleoside analogue to the mammal in the form of the nucleoside-lipidderivatives disclosed herein. Also disclosed is a method for avoiding orovercoming resistance of the retrovirus to nucleoside analogues throughadministering the analogue in the form of the lipid derivativecompositions disclosed herein.

Finally, the present invention includes use of these compositions in thepreparation of a medicament for treatment of a human viral infection.

Liposomal delivery of antiretroviral and antiviral drugs results inhigher dosing of macrophage and monocyte cells which take up liposomesreadily. The unique advantages of the present invention are that thelipid derivatives of the antiviral nucleosides are incorporatedpredominantly into the phospholipid layer of the liposome rather than inthe aqueous compartment. This allows larger quantities of antiviralanalogue to be incorporated in liposomes than is the case when watersoluble phosphate esters of the nucleosides are used. Completeincorporation of the antiviral derivative into liposomes will beobtained, thus improving both the drug to lipid ratio and the efficiencyof formulation. Further, there will be no leakage of the antiviral lipidanalogues from the liposome during storage. Finally, liposomal therapyusing these compositions allows larger amounts of antiviral compound tobe delivered to the infected macrophage and monocyte cells. Therapy withliposomal compositions containing site specific ligands allows stillgreater amounts of antiviral compounds to be delivered with increasedspecificity.

Another novel advantage of this invention is that each class of lipidderivatives of antiviral nucleosides disclosed below is believed to giverise directly to antiviral phosphorylated or non-phosphorylatednucleosides upon cellular metabolism.

A further advantage of this invention is that the novel lipidderivatives are incorporated into the cell, protecting the cell forprolonged periods of time, up to or exceeding 48 hours after the drug isremoved.

These and other advantages and features of the present invention willbecome more fully apparent from the following description and appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-5 are graphs plotting p24 production by HIV-infected cells as afunction of the amount of the composition of the present inventionadministered in vitro.

DETAILED DESCRIPTION OF THE INVENTION

The present invention involves lipid derivatives of nucleoside analogueswhich can be incorporated into the lipid bilayer of liposomes. Thesederivatives are converted into nucleoside analogues by constituentcellular metabolic processes, and have antiviral effects in vivo and invitro.

Suitable lipid derivatives of nucleoside analogues comprise phosphatidylnucleosides, nucleoside diphosphate diacylglycerols, nucleoside acylphosphates, and ceramide phosphonucleosides. With the exception of theacyl phosphates, which can include from one to five acyl groups, thelipid derivatives of these compositions provide one or two hydrophobicacyl groups to anchor the nucleoside in the lipid bilayer of theliposome. The present invention also comprises lipid derivatives capableof providing additional acyl groups, and hence greater anchoringstrength for nucleoside analogues. The increase in anchoring strengthmakes it possible to utilize nucleoside analogues of greater polarity inliposome formulations. Accordingly we disclose additional nucleosidestructures of this type for use in liposomal therapies. We also discloselipid derivatives of nucleoside analogues in which the lipid group isdirectly attached to the nucleoside, rather than through a phosphatelink.

Nomenclature

The lipid derivatives of the present invention are made up of complexstructures which can only be rigorously defined by cumbersometerminology. For purposes of clarity, the descriptions of lipid andnucleosides components and their combinations will be in terms ofcommonly used trivial names, familiar to those in the art. For example,the well known drug, 3′-azido-3′-deoxythymidine, will be frequentlyreferred to as AZT. Similarly the derivative of AZT comprising a 1,2diacylglycerol-3-phosphate moiety, will be frequently referred to asphosphatidylAZT or PAZT. Parallel derivatives of dideoxythymidine ordideoxycytidine will correspondingly be referred to as phosphatidylddTor pddT and phosphatidylddC and pddC. Derivatives of halogenatednucleosides will be referred to as, for example, phosphatidyl-3′BrddT.

The nucleoside analogues of the invention can be any nucleoside thatdoes not occur naturally in the species to be treated for viralinfection. It may comprise a naturally occurring purine or pyrimidinebase attached to an analogue of a naturally occurring ribose group. Itmay likewise comprise an analogue of a purine or pyrimidine baseattached to a ribose or deoxyribose group which is present in naturallyoccurring nucleosides. Alternatively, both the base and the ribosemoieties of the nucleoside analogues may be analogues of those found innature. A nucleoside analogue may also comprise either a normal base ora base analogue attached to a non-ribose sugar moiety.

Analogues of both the purine or pyrimidine base and the ribose group candiffer from a corresponding naturally occurring moiety by having newsubstituent groups attached thereto, by having naturally occurringsubstituent groups deleted therefrom, or by having atoms normallypresent replaced by others. Examples of analogues formed by substitutionare 2,6-diaminopurine and 3′-azido-3′deoxyribose; by deletion,6-oxypurine or didehydroribose; by replacement, 8-azaguanine.

Nucleoside analogues may also comprise a purine or pyrimidine baseattached to the pentose moiety in a non-naturally occurring linkage,such as, for example through the nitrogen at the 3 position rather thanthe 1 position of the pyrimidines.

In general, the nucleoside analogues used in preparing the liposomes ofthe present invention will have a purine or pyrimidine base, e.g.,adenine, guanine, cytosine or thymine, or an analogue thereof, attachedto a pentose, such as ribose or a ribose residue and/or derivative. Theattachment is through the nitrogen in the 9 position of the purines andthrough the nitrogen in the 1 position of the pyrimidines. Thesenitrogens are linked by a β-N-glycosyl linkage to carbon 1 of thepentose residue.

The pentose residue may be a complete pentose, or a derivative such as adeoxy- or dideoxypentose. In addition, the pentose residue can be afragment of a pentose, such as a hydroxylated 2-propoxymethyl residue ora hydroxylated ethoxymethyl residue. Particular nucleoside residueshaving these structures include acyclovir and gancyclovir. The pentosemay also have an oxygen or sulfur substitution for a carbon atom at, forexample, the 3′position of deoxyribose (BCH-189).

The phosphate groups are generally connected to the 5′ carbon of thepentoses in the compounds of the present invention; however, compoundswherein the phosphate groups are attached to the 3′ hydroxyl group ofthe pentose are within the invention if they possess antiviral activity.Where lipids are linked directly to pentose groups, those linkages mayalso be made either through the 3′ or preferably through the 5′ pentosecarbon.

It is important to recognize that in compounds having pentose residuesthat are not complete pentoses, the phosphate groups are connected tothe carbon that would have been the 5′ carbon if the pentose werecomplete. In these pentose fragments, the 2′ and/or 3′ carbons may bemissing; nevertheless, they are considered to be nucleoside derivativeswithin the meaning of present invention, and the carbon atom to whichthe phosphate groups are connected will generally be referred to hereinas the 5′ carbon for purposes of consistency of usage.

Any lipid derivative of a nucleoside analogue having an antiviralactivity is within the scope of the invention. The antiviral activitymay reside in any component of the lipid-nucleoside complex, that is, ina nucleoside base analogue, in a ribose analogue, or in the substitutionof another pentose for ribose. It may also reside in the complex as awhole, wherein, for example, a weakly antiviral analogue or onepossessing imperceptible or latent viral activity becomes more potentfollowing its incorporation into a lipid derivative of a nucleotide.

Nucleosides known to have such activity are members of the classcomprising 3′-azido-2′,3′-dideoxypyrimidine nucleosides, for example,AZT, AZT-P-AZT, AZT-P-ddA, AZT-P-ddI, AzddClU, AzddMeC, AzddMeC N4-OH,AzddMeC N4Me, AZT-P-CyE-ddA, AzddEtU(CS-85), AzddU(CS-87), AzddC(CS-91),AzddFC, AzddBrU, and AzddIU; the class comprising 3′-halopyrimidinedideoxynucleosides, for example, 3-FddClU, 3-FddU, 3-FddT, 3-FddBrU, and3-FddEtU; the class comprising 2′,3′-didehydro-2′,3′-dideoxynucleosides(D4 nucleosides), for example, D4T, D4C, D4MeC, and D4A; the classcomprising 2′,3′-unsubstituted dideoxypyrimidine nucleosides, forexample, 5-F-ddC, ddC and ddT; the class comprising 2′,3′-unsubstituteddideoxypurines nucleosides, for example, ddA, ddDAPR(diaminopurine),ddG, ddI, and ddMeA(N6 methyl); and the class comprisingsugar-substituted dideoxypurine nucleosides, for example, 3-N₃ddDAPR,3-N₃ddG, 3-FddDAPR, 3-FddG, 3-FddaraA, and 3-FddA, wherein Me is methyl,Et is ethyl and CyEt is cyanoethyl.

Other suitable nucleotide analogues may be antiviral agents likeacyclovir or gancyclovir (DHPG), or other analogues, as described below.Preferred dideoxy derivatives are those used in the treatment of AIDS,including 3′-azido-3′-deoxythymidine (azidothymidine or AZT);2′,3′-dideoxythymidine (ddT); 2′,3′-dideoxycytidine (ddC);2′,3′-dideoxyadenosine (ddA); and 2′,3′-dideoxyguanosine (ddG). AZT,ddT, and ddC are most preferred analogues at present. Thedidehydropyrimidines, as well as carbovir, a carbocyclic2′,3′-didehydroguanosine, are also preferred. The 3′-azido derivativesof deoxyguanosine (AZG) and the pyrimidine, deoxyuridine, and the3′-fluoro derivatives of deoxythymidine and deoxyguanosine are preferredas well. Among the 2′,6′-diaminopurines, the 2′,3′-deoxyriboside and its3′-fluoro and 3′-azido derivatives are preferred. Also preferred is2-chloro-deoxyadenosine.

Among the acyclic sugar derivatives,9-(4,-hydroxy-1′,2′-butadienyl)adenine (adenallene) and its cytosineequivalent are preferred. Preferred acyclic derivatives having a purineor diaminopurine base are 9-(2-phosphonylmethoxyethyl)adenine andphosphonomethoxyethyl deoxydiaminopurine (PMEDADP).

Stereoisomers of these nucleosides, such as 2′-fluoro-ara-ddA, may beadvantageous because of their resistance to acid-catalyzed hydrolysis ofthe glycosidic bond, which prolongs their antiviral activity. In suchcases, they are preferred.

For treating herpes, cytomegalovirus and hepatitis B infections, one mayutilize the lipid derivatives of acyclovir, gancyclovir,1-(2′-deoxy-2′-fluoro-1-β-D-arabinofuranosyl)-5-iodocytosine (FIAC) or1(2′-deoxy-2′-fluoro-1-β-D-arabinofuranosyl)-5-iodouracil (FIAU).

The lipids are preferably attached to the nucleoside analogues throughphosphate linkages. Lipid derivatives comprising a phosphate linkbetween a nucleoside analogue and lipid may be prepared fromphospholipids, phosphorylated nucleoside analogs, or both. Suitablephospholipids comprise phosphoglycerides, sphingolipids, or acylphosphates.

Lipid derivatives of nucleoside analogue in which lipids are linkedeither through mono-, di-, or triphosphate groups may be prepared fromphosphorylated nucleoside analogues. Phosphorylated nucleoside analoguesare known. The dideoxynucleoside analogue is phosphorylated according toconventional procedures such as the phosphorous oxychloride method ofToorchen and Topal (20). The preferred modified analogue is the5′-monophosphate. Since AZT, ddC and other dideoxynucleosides have onlythe 5′-hydroxyl, only the 5′-monophosphate is formed duringphosphorylation; however, in other analogues in which the 3′hydroxyl ispresent, a 3′-monophosphate can be formed. The diphosphate andtriphosphate analogues of antiviral nucleosides may also be used.

The aliphatic groups of the lipid moieties preferably have chain lengthsof two to twenty-four carbon atoms and have zero to six double bonds.The aliphatic groups may be attached to the glycerol moiety by acyl,ether or vinyl ether bonds.

Synthetic Methods

The lipid-nucleotide compounds of the present invention can besynthesized according to general methods applicable to all lipids andall antiviral nucleosides described below, as indicated in the flowdiagram of Figure and demonstrated specifically in Examples 1 through 7.

Lipids comprising fatty acids, alcohols, glycerides and phospholipidsmay be purchased from commercial suppliers (Avanti Polar Lipids, Inc.,Pelham, Ala. 35124) or may be synthesized according to known methods.Antiviral nucleoside analogues are available from Aldrich, Milwaukee,Wis. or from Sigma, St. Louis, Mo.

It is important that all traces of water be removed from the reactantsin order for the coupling reactions to proceed. Therefore, the lipidsare first either freeze-dried by solvent evaporation under vacuum, or ina vacuum oven over P₂O₅. The reactions are also carried out under aninert gas, such as, for example, argon.

The compounds of the invention can be formed according to syntheticprocedures which couple a phospholipid to a nucleoside analogue or whichcouple a phospholipid to a nucleoside analogue monophosphate ordiphosphate, wherein the phosphate group is located on the ribose groupof the nucleoside, at either the 3′ or preferably the 5′ location.

Lipids suitable for coupling to nucleosides, comprising primarily longchain fatty acids or alcohols, monoglycerides or diglycerides, ceramidesand other lipid species described below, may be phosphorylated bytreatment with appropriate agents, for example using phenylphosphorodichloridate according to the procedure of Brown (32), bytreatment with phosphorus oxychloride as in Example 6, or by other knownphosphorylation procedures.

In the first type of synthesis, a phospholipid, such as, for example, aphosphatidic acid, is coupled to a selected nucleoside analogue ateither the 3′ or 5′ hydroxyl by means of a coupling agent, such as, forexample, 2,4,6-triisopropylbenzenesulfonyl chloride in the presence of abasic catalyst, for example, anhydrous pyridine, at room temperature.Other coupling agents, such as dicyclohexylcarbodiimide can be used.

Lipid derivatives may also be synthesized by coupling a phosphatidicacid to an antiviral nucleoside monophosphate through a pyrophosphatebond. In this procedure, the nucleoside monophosphate or diphosphate isconverted to a derivative having a leaving group, for example,morpholine, attached to the terminal phosphate group, according to theprocedure of Agranoff and Suomi (21) and as illustrated in Example 4,for preparing a derivative of AZT and Example 6, for a derivative ofddA. A coupling of the phosphatidic acid and the nucleoside phosphatemorpholidate occurs on treatment of a dry mixture of the two reactantswith a basic catalyst, such as anhydrous pyridine, at room temperature.

The reactions are followed using thin layer chromatography (TLC) andappropriate solvents. When the reaction, as determined by TLC iscomplete, the product is extracted with an organic solvent and purifiedby chromatography on a support suitable for lipid separation, forexample, silicic acid.

The synthesis of products comprising adenine or cytidine having reactiveamino groups may be facilitated by blocking those groups with acetatebefore the coupling reaction by treatment with acetic anhydride; afterthe chromatography of the final product, the amino groups are unblockedusing ammonium hydroxide (Example 3).

Lipid Derivatives

Compounds which will be most effective will have a lipid portionsufficient to be able to incorporate the material in a stable way into aliposomal bilayer or other macromolecular array.

Some preferred lipid derivatives of nucleoside analogues that are withinthe scope of the present invention fall into four general classes:

1. Antiviral Phosphatidylnucleosides

The structure of these antiviral lipid compounds is shown below:

where N is a “chain terminating” dideoxynucleoside such as AZT, ddC,ddA, ddI, or another antiviral nucleoside such as acyclovir organcyclovir, A is a chalcogen (O, C or S), and R₁ and R₂, which may bethe same or different, are C₁ to C₂₄ aliphatic groups, having from 0 to6 sites of unsaturation, and preferably having the structureCH₃—(CH₂)_(a)—(CH═CH—CH₂)_(b)—(CH₂)_(c)—Y

wherein the sum of a and c is from 1 to 23; and b is 0 to 6; and whereinY is C(O)O⁻, C—O⁻, C═C—O⁻, C(O)S—, C—S—, C═C—S—, forming acyl ester,ether or vinyl ether bonds, respectively, between the aliphatic groupsand the glycerol moiety. These aliphatic groups in acyl ester linkagetherefore comprise naturally occurring saturated fatty acids, such aslauric, myristic, palmitic, stearic, arachidic and lignoceric, and thenaturally occurring unsaturated fatty acids palmitoleic, oleic,linoleic, linolenic and arachidonic. Preferred embodiments comprise amonoester or diester, or a 1-ether, 2-acyl ester phosphatidylderivative. In other embodiments, the aliphatic groups can be branchedchains of the same carbon number, and comprise primary or secondaryalkanol or alkoxy groups, cyclopropane groups, and internal etherlinkages.

This class of compounds may be prepared, for example, from the reactionof a diacylphosphatidic acid and an antiviral nucleoside analogue inpyridine as described for the preparation of 1,2dimyristoylglycerophospho-5′-(3′-azido-3′-deoxy)thymidine in Example 1.

Upon liposomal uptake, the compounds are believed to undergo metabolismby the phospholipases present in the cell. For example, in the specificcase of a diacylphosphatidyl derivative of a nucleoside, phospholipase Cwould act to give a diacylglycerol and the nucleoside monophosphate asshown below:

Alternatively, the same phosphatidylnucleoside may be hydrolyzed byphospholipase A and lysophospholipase followed by phosphodiesterase togive glycerol and nucleoside monophosphate by the sequence shown below:

2. Antiviral Nucleoside Diphosphate Diglycerides

The chemical structure of this class of compounds is shown below:

where N, A and R₁ and R₂ are as described above.

Nucleoside diphosphate diglycerides are known. The antiviral nucleosidediphosphate diglycerides may be prepared from phosphatidic acid and theantiviral nucleotide monophosphomorpholidates by the method of Agranoffand Suomi (21) as modified by Prottey and Hawthorne (22). This type ofsynthesis is presented in Example 4 for the synthesis of AZT5′-diphosphate dipalmitoyl glycerol.

Upon liposomal delivery to cells, this class of compounds will take partin several types of reactions since it is an analogue ofCDP-diglyceride, an important naturally-occurring intermediate in thebiosynthesis of phosphatidylglycerol, cardiolipin andphosphatidylinositol as shown below:

All of these reactions generate nucleoside monophosphate and a newphospholipid. It is important to note that Poorthuis and Hostetler (23)showed previously that a variety of nucleosides could substitute forCDP-diglyceride in these reactions, including UDP-diglycerideADP-diglyceride and GDP-diglyceride (23). Significantly, Ter Scheggett,et al. (24) synthesized deoxy CDP-diglyceride and found that it couldalso replace CDP-diglyceride in the mitochondrial synthesis ofphosphatidyiglycerol and cardiolipin, thereby suggesting the possibilityof using these novel compounds to generate the antiviral nucleosidephosphates in the target cells.

CDP-diglyceride hydrolase catalyzes another important metabolicconversion which gives rise to nucleoside monophosphate and phosphatidicacid, as shown below:

This pathway was first described in mammalian tissues by Rittenhouse, etal. (25). This enzyme, which is a pyrophosphatase, is expected to cleavedideoxynucleoside diphosphate diglyceride to the nucleosidemonophosphate and phosphatidic acid, providing a second manner in whichthe nucleoside monophosphate can be formed in the target cells.

3. Antiviral Nucleoside Acyl Phosphates

Another way to introduce a lipid compound into cells by means ofliposomes is to synthesize acyl esters of the nucleoside monophosphates,diphosphates or triphosphates. This synthesis may be carried outaccording to the procedure in Example 5 for the synthesis of dihexadecylphospho-5′-dideoxycytidine.

The structure of a diacylphosphonucleoside is shown below:

wherein N, A, R₁ and R₂ are as previously defined. In principle, one ormore acid moieties of the phosphate may be esterified and many othercombinations of phosphate and fatty alcohol substitution are possible.For example, a nucleoside monophosphate could have one or two aliphaticesters; a nucleoside diphosphate could have one to three aliphaticesters, and the nucleoside triphosphate could have one to four aliphaticesters. Nucleosides can be “chain terminating” dideoxynucleosides orother antiviral nucleosides.

Since cells contain a variety of esterases, it is anticipated that thisclass of compounds will be hydrolyzed to the phosphorylated nucleoside,bypassing the deficiency of dideoxynucleoside kinase in human monocytesand macrophages, and thereby restoring the antiviral activity.

4. Ceramide Antiviral Phosphonucleosides

Antiviral nucleoside phosphates can also be generated in cells afterliposomal delivery of ceramide antiviral nucleoside phosphates havingthe general structure shown below:

where CER is an N-acylsphingosine having the structure:

wherein R is as defined previously, or an equivalent lipid-substitutedderivative of sphingosine, and N is a “chain terminating” antiretroviralnucleoside or antiviral nucleoside as previously defined. This class ofcompounds is useful in liposomal formulation and therapy of AIDS andother viral diseases because it can be acted upon by sphingomyelinase orphosphodiesterases in cells giving rise to nucleoside monophosphate. Inaddition to the compound shown above, ceramide diphosphatedideoxynucleosides can also be synthesized, which may be degraded bycellular pyrophosphatases to give nucleoside monophosphate and ceramidephosphate.

Ceramide antiviral nucleoside phosphates may be prepared in a methodsimilar to the method for preparing antiviral nucleoside diphosphatediglycerides, with appropriate changes to the starting materials.

5. Other Lipid Derivatives of Antiviral Nucleosides

One approach to achieving even greater stability of lipid derivatives ofnucleoside analogues within liposomes is by increasing lipid-lipidinteraction between the lipid-nucleoside structure and the bilayer.Accordingly, in preferred embodiments, lipid derivatives of nucleosideanalogues having up to four lipophilic groups may be synthesized. Oneclass of these comprises diphosphatidylglycerol derivatives, having thegeneral structure:

In this class, nucleosides are attached to one or both phosphates by aphosphodiester bond to the 5′-OH of the deoxyribose, ribose ordideoxyribose moiety of the antiviral nucleoside. In the case of acyclicnucleosides, such as acyclovir or gancyclovir, the link would be to theOH group equivalent to that of the ribose, deoxyribose or dideoxyribose5′-position. There may be one or two nucleosides attached to eachmolecule. Nucleoside phosphates may also be attached by a pyrophosphatebond, as in Example 4.

Another class of derivatives having increased lipid components comprisesbis(diacylglycero)phosphonucleotides, having the general structure:

R₁₋₄ may be two, three or four aliphatic groups which are independentlyR as previously defined, said groups being in acyl ester, ether, orvinyl ether linkages. This compound may be made by the method of Example3.

The diphosphate version of this compound, with the following structure:

may be made by coupling the nucleoside monophosphomorpholidate to thephosphoester residue of bis(diacylglycero)phosphate according to theprocedure of Example 4. This compound will be metabolized to AZT-P inthe cells by CDP-diglyceride hydrolase (a pyrophosphatase). These twotypes of compounds may provide superior metabolic and physicalproperties.

Other suitable lipid derivatives of nucleosides may be synthesized usingnovel lipids. It is desirable, for example, to synthesize phospholipidderivatives of antiviral and antiretroviral nucleosides which will giverise to potent antiviral agents upon alternate paths of metabolism bythe target cells which take up the lipid formulation. For derivativesmade up of the following types of compounds, one might anticipate acellular metabolism distinct from that of more conventional phospholipidderivatives, because these have a phosphate group which is removed fromthe usual lipid group by a nitrogen containing group. The structure ofthese lipids features a quaternary ammonium derivative.

The compound shown:

D,L, -2,3-distearoyloxypropyl (dimethyl)-p-hydroxyethyl ammoniumacetate, was first synthesized by Rosenthal and Geyer in 1960 (35) andis available from Calbiochem, La Jolla, Calif. 92039. It can readilyserve to link AZT-phosphate or any other antiviral nucleoside phosphate,using triisopropylbenzenesulfonyl chloride (TIBSC) as described inExample 1 or 7.

Alternatively, AZT may be linked to the phosphorylated ammonium lipidprepared by POCl₃, using TIBSC. Shown below is the AZT derivative of thephosphorylated compound I,D,L,-2,3-diacyloxypropyl(dimethyl)-β-hydroxyethyl ammonium acetate,where R₁ and R₂ are aliphatic groups as previously defined, of thepreferred structure:

Further, the Compound I of Rosenthal and Geyer may also bephosphorylated as they describe in their paper (35). One may also usethe phosphorous oxychloride method of Toorchen and Topal (20) to preparethe phosphate ester of I. To this phosphorylated species one may thencouple any antiviral or antiretroviral nucleoside using the morpholidatederivative of the nucleoside phosphate as reported by Agranoff andSuomi, (21) and modified by Prottey and Hawthorne, 1967 (22). Theresulting nucleoside diphosphate derivatives of I may have exemplaryproperties as antiviral agents delivered in liposomes to infected cells.Preferred nucleosides include, but are not limited to: AZT, ddA, ddC,ddI, acyclovir, and gancyclovir. The AZT diphosphate derivative ofCompound I is shown below:

In any of the lipids derivatives described in the preceding sections 1through 5 above, the nucleoside may be any antiviral nucleoside; R₁₋₂(as well as R₃₋₄ for the bis(diacylglycero) species) may be anysaturated or unsaturated fatty acid having from 2 to 24 carbon atoms.Polyunsaturated, hydroxy, branched chain and cyclopropane fatty acidsare also possible. The stereochemistry of the glycerol moieties caninclude sn-1 or sn-3 phosphoester bonds or racemic mixtures thereof.There may be 1 or 2, (as well as 3, or 4 for the bis(diacylglycero)species) acyl ester groups, or alkyl ether or vinyl ether groups, asrequired.

A variety of other phospholipids may be linked to nucleosides,including, but not limited to phosphatidylglycerol,phosphatidylinositol, or any other phospholipid wherein the head groupcontains an available linking hydroxyl group, in either a naturalpolyhydroxyl alcohol such as inositol, or one in which it has beensubstituted by another polyhydroxy alcohol or by a carbohydrate, such asa sugar, again either natural or synthetic. In this case the nucleosidephosphate will be added by esterification to one or more of thehydroxyls of the alcohol or carbohydrate. Other glycolipids may alsoserve as the ligand to which the phosphate group of the nucleotide isattached by means of esterification to a glycolipid hydroxyl group.Other glycolipids, whether or not phospholipids, such as selectedcerebrosides or gangliosides, either natural or synthetic, havingsuitable hydrophobic properties may also be advantageously used. Thesemay also be linked to nucleotides by similar esterification ofcarbohydrate hydroxyl groups.

Furthermore, antiviral nucleosides may be linked to the phosphate groupsof the phosphatidylinositol mono-, di- and triphosphates, or to thephosphate-substituted carbohydrate moieties of phospholipids orglycolipids, either natural or synthetic.

Phosphatidylserine may be linked to nucleoside analogues directly byesterification of its carboxyl group with the 5′-hydroxyl of thenucleoside ribose group. Synthetic phospholipids which are similar instructure to phosphatidylserine, containing a carboxyl group in thepolar headgroup, may be linked in a similar way.

Phospholipids having alkyl chains attached by ether or vinyl ether bondsmay also be used to prepare nucleotide derivatives according to thepresent invention. Suitable phospholipids for this purpose comprisenaturally occurring acetal phosphatides, or plasmalogens, comprising along chain fatty acid group present in an unsaturated vinyl etherlinkage. Alternatively, analogs of 1-O-alkyl glycerol or 2-O-alkylglycerol may be prepared synthetically, and linked to a selectednucleotide as described in Example 7. Derivatives ofglycero-3-phospho-5′-azidothymidine are preferred, and may be preparedby condensing AZT monophosphate with various analogs of1-O-alkyl-glycerol having an alkyl group of 2 to 24 carbon chain lengthat the 1 position of glycerol. The 1-O-alkyl group may consist of asaturated, unsaturated aliphatic group having a chain length of 2 to 24carbon atoms. The 1-O-alkyl glycerol residue may be racemic orstereospecific. This compound may be acylated with fatty acid chloridesor anhydrides resulting in the synthesis of 1-O-alkyl,2-acyl-glycero-3-phospho-5′azidothymidine. Similarly, by using a largeexcess of azidothymidine monophosphate, the 1-O-alkyl,2,3-bis(phospho-5′-3′-azido, 3′-deoxythymidine)glycerol analogs may besynthesized. These derivatives have the general structure:

Where R₁ is an unsaturated or saturated alkyl chain 1 to 23 carbon atomsin length in ether or vinyl ether linkage. R₂ is OH or a saturated orunsaturated fatty acid ester of 2 to 24 carbon atoms. An ether or vinylether link at R₂ is also possible. The group at position 1 of glycerolmay also be OH if R₂ is the ether linked alkyl chain.N is any antiviralnucleoside linked in a 5′ phosphodiester link and A is a chalcogen (O, Cor S).

Although phosphorylated antiviral nucleosides (nucleotides) arepreferred embodiments of the present invention, it is possible toutilize non-phosphorus containing lipid derivatives of nucleosideanalogues if it is not necessary to provide the infected cell with thenucleoside phosphate in order to achieve an antiviral effect through theprocesses of cellular metabolism. Some examples of compounds of thistype would have fatty acids esterified, or present in alkyl linkage,directly to the 5′-hydroxyl of the nucleoside according to the syntheticmethod of Example 13.

Alternatively, a “spacer” molecule having, for example, carboxyl groupsat either end and 0 to 10 CH₂ groups in the center, could be esterifiedto the 5′-hydroxyl of the antiviral nucleoside. The other carboxyl ofthe “spacer” may be esterified to the free hydroxyl of diacylglycerol orany other lipid having an available hydroxyl function. Other linking(“spacer”) groups with suitable functional groups at the ends may alsobe used to link the diglyceride or other suitable lipid group to thenucleoside, by chemical methods well known to those skilled in the art.

Preparation of Liposomes Comprising Lipid Derivatives of AntiviralNucleosides

After synthesis, the lipid derivative of the nucleoside analogue isincorporated into liposomes, or other suitable carrier. Theincorporation can be carried out according to well known liposomepreparation procedures, such as sonication, extrusion, ormicrofluidization. Suitable conventional methods of liposome preparationinclude, but are not limited to, those disclosed by Bangham, et al. (4),Olson, et al. (26), Szoka and Papahadjapoulos (27), Mayhew, et al. (28),Kim, et al. (29), Mayer, et al. (30) and Fukunaga, et al. (31).

The liposomes can be made from the lipid derivatives of nucleosideanalogues alone or in combination with any of the conventional syntheticor natural phospholipid liposome materials including phospholipids fromnatural sources such as egg, plant or animal sources such asphosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol,sphingomyelin, phosphatidylserine, or phosphatidylinositol. Syntheticphospholipids that may also be used, include, but are not limited to,dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidycholine, and thecorresponding synthetic phosphatidylethanolamines andphosphatidylglycerols. Other additives such as cholesterol or othersterols, cholesterol hemisuccinate, glycolipids, cerebrosides, fattyacids, gangliosides, sphingolipids, 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP), N-[1-(2,3-dioleoyl)propyl]-N,N,N-trimethylammonium (chloride) (DOTMA),D,L,-2,3-distearoyloxypropyl(dimethyl)-β-hydroxyethyl ammonium(acetate), glucopsychosine, or psychosine can also be added, as isconventionally known. The relative amounts of phospholipid and additivesused in the liposomes may be varied if desired. The preferred ranges arefrom about 80 to 95 mole percent phospholipid and 5 to 20 mole percentpsychosine or other additive. Cholesterol, cholesterol hemisuccinate,fatty acids or DOTAP may be used in amounts ranging from 0 to 50 molepercent. The amounts of antiviral nucleoside analogue incorporated intothe lipid layer of liposomes can be varied with the concentration oftheir lipids ranging from about 0.01 to about 100 mole percent.

Using conventional methods to entrap active compound entrapsapproximately 20 to 50% of the material present in solution; thus,approximately 50 to 80% of the active compound is wasted. In contrast,where the nucleoside analogue is incorporated into the lipids, virtuallyall of the nucleoside analogue is incorporated into the liposome, andvirtually none of the active compound is wasted.

The liposomes with the above formulations may be made still morespecific for their intended targets with the incorporation of monoclonalantibodies or other ligands specific for a target. For example,monoclonal antibodies to the CD4 (T4) receptor may be incorporated intothe liposome by linkage to phosphatidylethanolamine (PE) incorporatedinto the liposome by the method of Leserman, et al. (19). As previouslydescribed, HIV will infect those cells bearing the CD4 (T4) receptor.Use of this CD4-targeted immunoliposome will, therefore, focus antiviralcompound at sites which HIV might infect. Substituting another CD4recognition protein will accomplish the same result. On the other hand,substituting monoclonal antibody to gp110 or gp41 (HIV viral coatproteins) will focus antiviral immunoliposomes at sites of currentlyactive HIV infection and replication. Monoclonal antibodies to otherviruses, such as Herpes simplex or cytomegalovirus will focus activecompound at sites of infection of these viruses.

Therapeutic Uses of Lipid Derivatives

The liposome incorporated phosphorylated nucleoside analogue isadministered to patients by any of the known procedures utilized foradministering liposomes. The liposomes can be administeredintravenously, intraperitoneally, intramuscularly, or subcutaneously asa buffered aqueous solution. Any pharmaceutically acceptable aqueousbuffer or other vehicle may be utilized so long as it does not destroythe liposome structure or the activity of the lipid nucleoside analogue.One suitable aqueous buffer is 150 mM NaCl containing 5 mM sodiumphosphate with a pH of about 7.4 or other physiological buffered saltsolutions.

The dosage for a mammal, including a human, may vary depending upon theextent and severity of the infection and the activity of theadministered compound. Dosage levels for nucleoside analogues are wellestablished. Dosage levels of lipid derivatives of nucleoside analoguesshould be such that about 0.001 mg/kilogram to 1000 mg/kilogram isadministered to the patient on a daily basis and more preferably fromabout 0.05 mg/kilogram to about 100 mg/kilogram.

The present invention utilizes the antiviral nucleoside derivativesnoted above incorporated in liposomes in order to direct these compoundsto macrophages, monocytes and any other cells which take up theliposomal composition. Ligands may also be incorporated to further focusthe specificity of the liposomes.

The derivatives described have several unique and novel advantages overthe water soluble dideoxynucleoside phosphates described in an earliercopending application.

First, they can be formulated more efficiently. Liposomes comprisinglipid derivatives of nucleoside analogues have much higher ratios ofdrug to lipid because they are incorporated into the wall of theliposome instead of being located in the aqueous core compartment.Secondly, the liposomes containing the lipophilic dideoxynucleosidederivatives noted above do not leak during storage, providing improvedproduct stability. Furthermore, these compositions may be lyophilized,stored dry at room temperature, and reconstituted for use, providingimproved shelf life. They also permit efficient incorporation ofantiviral compounds into liposomal formulations without significantwaste of active compound.

They also provide therapeutic advantages. Stability of the liposomallyincorporated agent causes a larger percentage of the administeredantiviral nucleoside to reach the intended target, while the amountbeing taken up by cells in general is minimal, thereby decreasing thetoxic side effects of the nucleosides. The toxic side effects of thenucleosides may be further reduced by targeting the liposomes in whichthey are contained to actual or potential sites of infection byincorporating ligands specifically binding thereto into the liposomes.

Finally, the compounds noted above have been constructed in a novel wayso as to give rise to phosphorylated dideoxynucleosides or otherantiviral nucleosides upon further cellular metabolism. This improvestheir antiretroviral (antiviral) effect in monocytes and macrophages orother cells which are known to be resistant to the effects of the freeantiviral compounds. Further, the compounds pre-incubated with lymphoidcells provide complete protection from HIV infection for up to andexceeding 48 hours after the drug is removed, while the free nucleosideprovides no protection 24 hours after removal. Finally, the lipidcompounds are expected to be useful in treating HIV infections due tostrains of virus which are resistant to free antiretroviral nucleosideanalogues.

Lipid derivatives of antiviral agents have a prolonged antiviral effectas compared to the lipid-free agents; therefore they provide therapeuticadvantages as medicaments even when not incorporated into liposomes.Non-liposomal lipid derivatives of antiviral nucleoside analogues may beapplied to the skin or mucosa or into the interior of the body, forexample orally, intratracheally or otherwise by the pulmonary route,enterally, rectally, nasally, vaginally, lingually, intravenously,intraarterially, intramuscularly, intraperitoneally, intradermally, orsubcutaneously. The present pharmaceutical preparations can contain theactive agent alone, or can contain further pharmaceutically valuablesubstances. They can further comprise a pharmaceutically acceptablecarrier.

Pharmaceutical preparations containing lipid derivatives of antiviralnucleosides are produced by conventional dissolving and lyophilizingprocesses to contain from approximately 0.1% to 100%, preferably fromapproximately 1% to 50% of the active ingredient. They can be preparedas ointments, salves, tablets, capsules, powders or sprays, togetherwith effective excipients, vehicles, diluents, fragrances or flavor tomake palatable or pleasing to use.

Formulations for oral ingestion are in the form of tablets, capsules,pills, ampoules of powdered active agent, or oily or aqueous suspensionsor solutions. Tablets or other non-liquid oral compositions may containacceptable excipients, known to the art for the manufacture ofpharmaceutical compositions, comprising diluents, such as lactose orcalcium carbonate; binding agents such as gelatin or starch; and one ormore agents selected from the group consisting of sweetening agents,flavoring agents, coloring or preserving agents to provide a palatablepreparation. Moreover, such oral preparations may be coated by knowntechniques to further delay disintegration and absorption in theintestinal tract.

Aqueous suspensions may contain the active ingredient in admixture withpharmacologically acceptable excipients, comprising suspending agents,such as methyl cellulose; and wetting agents, such as lecithin orlong-chain fatty alcohols. The said aqueous suspensions may also containpreservatives, coloring agents, flavoring agents and sweetening agentsin accordance with industry standards.

Preparations for topical and local application comprise aerosol sprays,lotions, gels and ointments in pharmaceutically appropriate vehicleswhich may comprise lower aliphatic alcohols, polyglycols such asglycerol, polyethylene glycol, esters of fatty acids, oils and fats, andsilicones. The preparations may further comprise antioxidants, such asascorbic acid or tocopherol, and preservatives, such as p-hydroxybenzoicacid esters.

Parenteral preparations comprise particularly sterile or sterilizedproducts. Injectable compositions may be provided containing the activecompound and any of the well known injectable carriers. These maycontain salts for regulating the osmotic pressure.

The therapeutically effective amount of the lipid derivatives isdetermined by reference to the recommended dosages of the activeantiviral nucleotide, bearing in mind that, in selecting the appropriatedosage in any specific case, consideration must be given to thepatient's weight, general health, metabolism, age and other factorswhich influence response to the drug. The parenteral dosage will beappropriately an order of magnitude lower than the oral dose.

A more complete understanding of the invention can be obtained byreferring to the following illustrative examples, which are notintended, however, to unduly limit the invention.

EXAMPLE 1 Synthesis of1,2-Dimyristoylglycerophospho-5′-(3′-azido-3′-deoxy)thymidine,monosodium salt Preparation of Dimyristoylphosphatidic Acid (DMPA-H)

In a separatory funnel (500 ml), dimyristoylphosphatidic acid disodiumsalt (1 g., 1.57 mmol) was first dissolved in chloroform:methanol (2:1by volume, 250 ml) and mixed well. Distilled water (50 ml) was added tothe solution, and the pH was adjusted to 1 by adding concentratedhydrochloric acid. The solution was mixed well and the chloroform layercollected. The chloroform layer was back washed once with methanol:water(1:1 by volume, 80 ml) and evaporated under reduced pressure at 30° C.to yield dimyristoylphosphatidic acid (DMPA-H) as a white foam.Cyclohexane (10 ml) was added and the solution lyophilized to dryness toobtain a white powder (850 mg) which was then stored at −20° C. A daybefore the coupling reaction, DMPA-H (250 mg, 0.42 mmol) was dissolvedin cyclohexane (10 ml) in a round-bottom (50 ml) flask and the solventevaporated under reduced pressure at room temperature. This process wasrepeated four more times and the DMPA-H further dried in the vacuum ovenat room temperature overnight over P₂O₅ and stored in a desiccator at−20° C.

Coupling Reaction

Under argon, to the 50 ml round-bottom flask containing dried DMPA-H(250 mg, 0.42 mmol), dried 3′-azido-3′-deoxythymidine (AZT), SigmaChemical, St. Louis, Mo., (85 mg, 0.31 mmol, dried over P₂O₅ undervacuum overnight), and 2,4,6-triisopropylbenzenesulfonyl chloride (315mg, 1.04 mmol) was added, and anhydrous pyridine (2 ml) added viasyringe to obtain a clear solution. The reaction mixture was stirred atroom temperature for 18 hours. (The reaction was followed by thin layerchromatography). Water (1 ml) was added to the crude product to destroyexcess catalyst and the solvent was evaporated under reduced pressure toyield a yellow gum which was then redissolved in a small volume ofmethanol:chloroform (1:9 by volume) and applied to a column of silicagel (45 g, Kieselgel 60, West Germany). The column was eluted with 8%methanol in chloroform. After a forerun (rejected), AZT was recovered,and then dimyristoylphosphatidyl-3′-azido-3′-deoxythymidine (DMPA-AZT)was obtained. The fractions containing the product were combined and thesolvent was evaporated under reduced pressure. Cyclohexane (5 ml) wasadded to the residue and the mixture lyophilized to dryness under vacuumover P₂O₅ to yield pure DMPA-AZT (270 mg, 0.29 mmol, 95%).

Conversion to Monosodium Salt

To the dried DMPA-AZT redissolved in chloroform:methanol (2:1 by volume,30 ml), distilled water (6 ml) was added, mixed well, and the pH of theaqueous layer was adjusted to 1. The chloroform layer was collected and10 ml of methanol:water (1:1,) was added and mixed well. The pH of theaqueous layer was adjusted to 6.8 with methanolic NaOH (0.1N N), mixedwell, and the aqueous layer was maintained at pH 6.8. The combinedchloroform, methanol and water mixture was evaporated under reducedpressure to yield dimyristoylphosphatidyl 3′-azido-3′-deoxythymidinemonosodium salt. The residue was redissolved in chloroform:methanol (2:1by volume, 2 ml) and acetone added to precipitate DMPA-AZT monosodiumsalt which was further dried from cyclohexane (5 ml) to yield a whitepowder (220 mg, 0.26 mmol, 78% yield based on AZT). The melting pointwas 230° C.; Rf value on silica gel G thin layer plates was 0.32(chloroform:methanol:water:ammonia 80:20:1:1), Rf 0.58(chloroform:methanol:water:ammonia 70:30:3:2), Rf 0.31(chloroform:methanol:water 65:25:4); UV absorption maximum 266 nm (ε10,800); Analysis Calculated for C₄₁N₅O₁₁P₁H₇₂. 1H₂O: C,57.24; H,8.44;P,3.61; Found: C,56.80; H,8.83; P,3.52. MS, m/e 864.60 (M+)

Proton NMR: (CDCL3) δ 0.88 (6H, bt, J=6.9 Hz, acyl CH3), 1.26 (40H, s,acyl CH2), 1.60 (4H, bs, β acyl CH2), 1.94 (3H, s, thymine CH3), 2.31(4H, m, ∝ acyl CH2), 2.39 (2H, m, ribose 2′H), 3.38 (2H, bd, J=12.6 Hz,ribose 5′H), 3.78 (2H, m, sn-3 CH₂ glycerol), 4.00 (1H, dd, J1=12 Hz,J2=6 Hz, sn-1 CH₂ glycerol), 4.18 (1H, dd, J1=12 Hz, J2=6 Hz, sn-1 CH₂glycerol), 4.07 (1H, m, ribose 3′H), 4.41 (1H, m, ribose 4′H), 5.24 (1H,m, sn-2 CH glycerol), 7.62 (1H, s, thymine 6H), 6.21 (1H, t, J=6 Hz,ribose 1′H). The peak area ratio of phosphatidic acid to AZT is 1.

EXAMPLE 2 Synthesis of1,1,2-Dimyristoylglycerophospho-5′-(3′deoxy)thymidine, monosodium salt

3′-deoxythymidine was obtained from Sigma Chemical, St. Louis, Mo. Thelipid derivative of this analogue was synthesized using the same methoddescribed above in Example 1. Melting Point 235° C., Rf on silica gel G0.25 (chloroform/methanol/water/ammonia 80:20:1:1); 0.57(chloroform:methanol:ammonia:water 70:30:3:2); 0.24(chloroform:methanol:water 64:25:4); UV absorption maximum 269 nm (ε8,400); Analysis: Calculated for C₄₁N₂O₁₁P₁H₇₂Na₁.1H₂O: C,58.53; H,8.87;P, 3.69; Found: C,56.75; H,9.33; P,3.58. MS, m/e 823.00 (M+).

Proton NMR: (CDCL3) δ 0.91 (6H, bt, J=6.8 Hz, acyl CH3), 1.23 (4H, bs,acyl CH2), 1.26 (4H, bs, acyl CH2), 1.28 (32H, bs, acyl CH2), 1.62 (4H,m, β acyl CH2), 1.97 (3H, s, thymine CH3), 2.05 (2H, m, ribose 2′H),2.35 (4H, m, ∝ acyl CH2), 3.39 (2H, bs, ribose 5′H), 3.90 (2H, m, sn-1CH₂ glycerol), 4.16 (1H, m, sn-1 CH₂ glycerol), 4.24 (1H, m, sn-1 CH₂glycerol), 4.38 (1H, m, ribose 4′H), 5.23 (1H, m, sn-2 glycerol) 6.10(1H, bt, ribose 1′H), 7.68 (1H, s, thymine 6H). The peak area ratio ofphosphatidic acid to 2′3′-dideoxythymidine is 1.

EXAMPLE 3 Synthesis of1,2-Dimyristoylglycerophospho-5′-(2′,3′-dideoxy)cytidine Preparation of4-acetyl-2′3′-dideoxycytidine

To a stirred, refluxing solution of 2′-3′-dideoxycytidine (DDC) (400 mg,1.89 mmol) in anhydrous ethanol (35 ml, dried first with Lindy type 4×molecular sieve, and twice distilled over magnesium turnings) was addedacetic anhydride (0.4 ml, 5.4 mmol). During the course of a 3 hourrefluxing period, four more additional 0.4 ml portions of aceticanhydride were added at 30 minute intervals. The reaction was followedby thin layer chromatography (silica gel F254, Kodak Chromagram,developed with 10% methanol in chloroform). After the final addition,the solution was refluxed for 1 more hour. The reaction mixture wascooled and solvent was evaporated under diminished pressure. The residuewas redissolved in 8% methanol in chloroform (5 ml) and chromatographedon a silica gel column (2.2 cm×30 cm, Kieselgel 60, 70-230 mesh, EMScience, 45 g). The column was eluted with 8% methanol in chloroform toyield pure 4-acetyl-2′3′-dideoxycytidine (DDC-OAC) in 80% yield.

Coupling Reaction

A day before the coupling reaction, DMPA-H (prepared as before, 250 mg,0.42 mmol) was dissolved in cyclohexane (10 ml) in a round-bottom flask(50 ml) and the solvent evaporated under reduced pressure at roomtemperature. This process was repeated four more times and DMPA-Hfurther dried in a vacuum oven at room temperature overnight over P₂O₅.Under argon, to the 50 ml round-bottom flask containing dried DMPA-H wasadded dried (DDC-OAC) (85 mg, 0.33 mmol, dried over P₂O₅ under vacuumovernight), and 2,4,6-triisopropylbenzenesulfonyl chloride (315 mg, 1.04mmol), and anhydrous pyridine (2 ml) via syringe to obtain a clearsolution. The reaction mixture was stirred at room temperature for 18hours. (The reaction was followed by thin layer chromatography). Water(1 ml) was added to the mixture to destroy excess catalyst. The solventwas evaporated under reduced pressure to yield a yellow gum which wasredissolved in a small volume of methanol in chloroform (1:9 by volume)and applied to a column of silica gel (45 g, Kieselgel 60, EM Science).The column was topped with a small amount of sand (500 mg) to preventthe sample from floating during elution. The column was eluted with 8%methanol in chloroform (1.5L). After a forerun (rejected), thendimyristoylphosphatidyl-5′-(2′3′-dideoxy)cytidine (DMPA-DDC) wasobtained. The fractions containing the product were combined and thesolvent was evaporated under reduced pressure. The residue was furtherdried with cyclohexane to yield pure DMPA-DDC-OAC (210 mg, 0.21 mmol, in70% yield). R_(f) 0.40 (silica gel GF, 20×20 cm, Analtech,chloroform:methanol:water: ammonia 80:20:1:1 by volume).

Deblocking with 9N NH4OH

DDC-OAC-DMPA (40 mg, 0.04 mmol) was dissolved in chloroform:methanol(1:1, 2 ml), and 9N NH₄0H (10 drops) was added at once. The solution wasstirred at room temperature for 15 minutes and was then quicklyneutralized with glacial acetic acid to pH 7. The neutralized solutionwas evaporated to dryness overnight under reduced pressure to yielddimyristoylphosphatidyl 5′(2′3′-dideoxy)cytidine (DMPA-DDC, 35 mg, 0.037mmol). Melting point: DMPA-ddC decomposed at 240° C. On thin layerchromatography on silica gel GF plates, the Rf values were: 0.11(chloroform:methanol:water:ammonia 80:20:1:1); 0.38(chloroform:methanol:ammonia:water 70:30:3:2); 0.15(chloroform:methanol:water 65:25:4); UV absorption maximum 273 nm (ε5,800).

NMR: (CDCL3) δ 0.86 (6H, by, acyl CH3), 1.24 (40H, bs, acyl CH2), 1.57(4H, m, β acyl CH2), 2.28 (4H, m, ∝ acyl CH2), 3.36 (2H, m, ribose 5′H),3.94 (2H, bs, sn-3 CH₂ glycerol), 4.19 (1H, m, sn-1 CH₂ glycerol), 4.29(1H, m, sn-1 CH₂ glycerol), 4.40 (1H, bs, ribose 4′H), 5.19 (1H, m, sn-2CH glycerol), 5.89 (1H, m, thymine 5-H), 7.44 (1H, bs, thymine NH3),7.94 (1H, bs, thymine NH₂). The peak area ratio of phosphatidic acid to2′3′-dideoxycytidine is 1.

EXAMPLE 4 Synthesis of (3′Azido-3′-deoxy)thymidine-5′-diphosphate-sn-3-(1,2-dipalmitoyl)glycerol Synthesis ofAZT-monophosphate Morpholidate

This compound was synthesized following the method of Agranoff and Suomi(21). AZT-monophosphate was converted into the acidic form by passing asolution in water through a column of Dowex 50W (50×2-200, 100-200 mesh,Sigma Chemicals, St. Louis, Mo.). A solution of 117 mg AZT-monophosphate(0.3 millimoles) in 3 ml of water was transferred to a two neck roundbottom flask. The 3 ml of t-butanol and 0.106 ml of freshly distilledmorpholine (1.20 millimoles) were added and the mixture was placed in aoil bath at 90° C. Four equivalents of dicyclohexylcarbodiimide 249 mg,1.20 millimole) in 4.5 ml of t-butanol were added dropwise. The reactionwas monitored by thin layer chromatography on silica gel 60, F 254,plates (E. Merck, Darmstadt) with chloroform/methanol/acetic acid/water(50/25/3/7 by volume) as the developing solvent. The reaction was notedto be complete after 3 hours. The mixture was cooled and after additionof 4.5 ml of water was extracted four times with 15 ml of diethylether.The aqueous layer was evaporated to dryness and dried in vacuo overP₂O₅. The product was obtained (199 mg, 100% yield) and used forcoupling to phosphatidic acid without further purification.

Coupling of AZT-monophosphate Morpholidate to DipalmitoylphospatidicAcid

Dipalmitoylphosphatidic acid, disodium salt was converted to the freeacid by extracting the material from chloroform by the method of Blighand Dyer (34) using 0.1N HCl as the aqueous phase. The chloroform layerwas evaporated to dryness in vacuo, the phosphatidic acid (196 mg, 0.3millimoles) was redissolved in chloroform and transferred to the vesselcontaining the AZT-monophosphate morpholidate. After the chloroform wasremoved in vacuo using a rotary evaporator, the mixture was dried byaddition and evaporation of benzene and finally dried in vacuo overP₂O₅. The reaction was started by addition of 30 ml of anhydrouspyridine and the clear mixture was stirred at room temperature. Thereaction was monitored with thin layer chromatography as noted abovewith chloroform/methanol ammonia/water (70/38/82 by volume) asdeveloping solvent. The Rf of phosphatidic acid, AZT-monophosphatemorpholidate and AZT-diphosphate dipalmitoylglycerol is 0.11, 0.50, and0.30, respectively.

After 70 hours the pyridine was evaporated and the product was extractedinto chloroform after addition of 15 ml of water, 30 ml of methanol, 22ml chloroform and sufficient 1N formic acid to adjust the pH to 4.0. Thecombined chloroform layers after two extractions were evaporated todryness, the residue was dissolved in chloroform/methanol/ammonia/water,70/38/8/2, and the product was purified by silica gel columnchromatography in this solvent applying an air pressure equivalent toone meter of water. Fractions not completely pure were further purifiedby HPLC on a reverse phase column (vydac C18) using water/methanol (8/2by volume) and methanol as eluents. Fractions containing the desiredproduct were evaporated to dryness to give 132 mg. of a white solid (44%yield) which gave a single spot by thin layer chromatography with silicagel g plates developed with chloroform/methanol/ammonia/water, 70/38/8/2(Rf 0.35) and chloroform/methanol/water, 65/35/4 (Rf 0.54).

500 MHz NMR (CDCl₃) δ 0.88 (3H, t, J=6.93 Hz, sn-2-acyl CH₃), 0.92 (3H,t, J=7.48 Hz, sn-1-acyl chain CH₃), 1.25 (48H, s, CH₂ acyl chains), 1.55(4H, bs, β CH₂ acyl chains) 1.83 (3H, s, CH₃ thymine), 2.25 (2H, t,J=6.97 Hz, 2H, alpha CH₂ sn-2-acyl chain), 2.27 (2H, t, J=7.79 Hz, ∝ CH₂sn-1-acyl chain), 2.44 (4H, bs, 2′ and 5′ H ribose), 3.78 (1H, dd,J=1.68, 5.51 Hz, 3′H ribose), 3.95 (2H, bs, sn-3 CH₂ glycerol), 4.07(1H, bs, He/_(a) sn-1 CH₂ glycerol), 4.13 (1H, bs, 1H, sn-2 CHglycerol), 4.36 (1H, bs, H/_(a)/H_(e) sn-1 CH₂ glycerol), 5.21 (1H, bs,sn-2 CH glycerol), 5.66 (1H, bs, 1′H ribose), 7.14 (1H, d, J=6.25 Hz, 6Hthymine). The ratio of acyl chains: glycerol:ribose: thymine as deducedfrom appropriate resonances amounted to 2.12:0.93:0.98:1.00. IR (KBr,disk) showed 2105 (azido), 1745 (c=o ester) and 1705 (c=o thymine) asidentifiable bands.

EXAMPLE 5 Synthesis of an Antiviral Nucleoside Diacyl Phosphate

Dihexadecyl phospho-5′-dideoxycytidine is synthesized according to themethod described in Example 1, except that the reactants aredideoxycytidine and dihexadecyl hydrogen phosphate. The startingmaterial dihexadecyl hydrogen phosphate is synthesized fromhexadecan-1-ol and phenyl phosphorodichloridate as first reported by D.A. Brown, et al. (32).

EXAMPLE 6 Synthesis of Dideoxyadenosine Diphosphate Ceramide anAntiviral Phosphonucleoside

The method of Example 2 is repeated, except that dideoxyadenosinemonophosphate morpholidate is substituted for the dideoxycytidinemonophosphate morpholidate. Ceramide phosphoric acid is prepared by theaction of phosphorus oxychloride on ceramide. Ceramide phosphoric acidis substituted for the dimyristoyl phosphatidic acid. Similar resultsare obtained.

EXAMPLE 7 Synthesis of 1-O-stearoylglycero-rac-3-phospho-5′-(3′-deoxy,3,-azido)thymidine

Dry 1-O-stearoyl-rac-3-glycerol (batyl alcohol, 250 mg),3′-azido-3′deoxythymidine monophosphate sodium salt (0.725 gm) and2,4,6,-triisopropylbenzenesulfonyl chloride (TPS, 1.219 gm) were mixedin dry pyridine and stirred overnight under nitrogen. Chloroform (50 ml)was added and the reaction mixture was washed twice with cold 0.2N HCland 0.2N sodium bicarbonate. The organic phase was removed in vacuo witha rotary evaporator and the product was crystallized at −20° C. from 20ml of chloroform/acetone (12:8 by volume). The final purification of thecompound was done by preparative thin layer chromatography using 500micron layers of silica gel G developed withchloroform/methanol/concentrated ammonia/water (70/30/1/1 by volume).

In the preceding syntheses, proton NMR spectra were obtained with aGeneral Electric QE-300 spectrometer, using tetramethylsilane asinternal standard (key: s=singlet, d=doublet, t=triplet, q=quartet,dd=doublet of doublets, b=broad), UV spectra were recorded on ShimadzuUV-160, spectrophotometer. Fast atom bombardment mass spectra weredetermined by Mass Spectrometry Service Laboratory, University ofMinnesota. Elemental analyses were determined by Galbraith Laboratories,Knoxyille, Tenn. and Schawarzkopf Microanalytical Laboratory, N.Y.Melting points were obtained with a Fisher-Johns melting apparatus.Column chromatography was carried out on Merck silica gel 60 (70-230mesh). Rf values were obtained with HPTLC Merck, Kieselgel 60 pre-coatedplates, 10×10 cm. Anhydrous pyridine, 2,4,6-Triisopropylbenzenesulfonylchloride (TPS), and 3′-azido-3′-deoxythymidine (AZT) were purchased fromAldrich. Dimyristoylphosphatidic acid, disodium salt, was purchased fromAvanti; batyl alcohol was obtained from Sigma Chemical, St. Louis, Mo.

EXAMPLE 8 Preparation of Liposomes Containing AntiretroviralLiponucleotides

6.42 micromoles of dioleoylphosphatidylcholine, 3.85 micromoles ofcholesterol, 1.28 micromoles of dioleoylphosphatidylglycerol and 1.28micromoles of dimyristoylphosphatidyl-azidothymidine were mixed in asterile 2.0 ml glass vial and the solvent was removed in vacuo in arotary evaporator. In some experiments,dimyristoylphosphatidylazidothymidine was replaced with eitherdimyristoylphosphatidyldideoxythymidine,dimyristoylphosphatidyldideoxycytidine or azidothymidine diphosphatedimyristoylglycerol; control liposomes were prepared by omitting theantiviral liponucleotide. The dried film was placed under high vacuumovernight at room temperature to remove traces of solvent. The lipidfilm was hydrated at 30° C. with 0.3 ml of sterile 10 mM sodium acetatebuffer (pH 5.0) containing isotonic dextrose and the ampule was sealed.The mixture was vortexed intermittently for 10 minutes followed bysonication using a Heat Systems Ultrasonics sonicator with a cup horngenerator (431B) at output control setting #9 for 90 to 120 minutes atwhich time the sample is clarified. This sonicated preparation wasdiluted with sterile RPMI buffer and added to the tissue culture wellsat the concentration indicated.

EXAMPLE 9 Coupling of Monoclonal Antibodies to CD4 to an AntiviralLipid-containing Liposome

Dimyristoylphosphatidyl-AZT produced by the method of Example 1,dimyristoylphosphatidylcholine, cholesterol anddimyristoylphosphatidylethanolamine in a molar ratio of 39:39:20:2. 200mg of this lipid mixture was dried in vacuo using a rotary evaporator toform a thin film in a 100 ml round-bottom flask. 1 ml of sterilephosphate buffered saline was added and the mixture shaken gently at 20°C. for 20 minutes, followed by ten 30-second cycles of vortexing to formmultilamellar liposomes. The suspension was subjected to 5 cycles ofextrusion through two stacked Nucleopore polycarbonate filters havingpore diameters of 200 nm to produce a homogeneous liposomal population.Other methods may be used such as sonication, reverse phase evaporationand use of a French press or Microfluidizer (Microfluidics, Newton,Mass.) 0.1 to 2 mg of OKT4a monoclonal antibodies to CD4 antigen arethiolated by incubation with 0.08 mM N-succinimidyl3-(2-pyridyldithio)propionate (SPDP). Untreated SPDP is removed by gelfiltration through Sephadex G25. The voiding DTP-protein is reduced with0.05 M dithiothreitol in 0.1 M acetate buffered saline at pH 4.5 for 20minutes, producing reduced thiolated antibody.

Liposomes produced by the method of Example 5, representing 5 micromolesof phospholipid are incubated overnight at room temperature with 1 mg ofthiolated antibody in 0.20 ml of isotonic MES/HEPES buffer, pH 6.7. Theresulting immunoliposomes are purified by the discontinuous metrizimidegradient method of Heath et al. (33) and sterilized by passage through200 nm filters.

EXAMPLE 10 Inhibition of HIV Replication in Tissue Culture Cells byLipid Nucleoside Conjugates

A. Methods

Viral Infection of Human T-cells

The human T lymphoblastoid cell line, CCRG-CEM (hereafter referred to asCEM), was grown in RPMI 1640 medium containing 100 U/ml penicillin G,100 ug/ml streptomycin, 2 mM glutamine and 10% fetal bovine serum(Hyclone Laboratories, Logan, Utah). Cells were infected with the LAV-1strain (L. Montagnier, Paris, France) at a multiplicity of infection ofone tissue culture 50% infectious dose (TCID₅₀)/cell for 60 minutes at37° C. in medium containing 1% polybrene. CEM cells were infected insuspension at 6×10⁴ cells/ml, washed three times by centrifugation andresuspension and then distributed in 96-well plates at 6×10⁴ cells/wellbefore addition of medium containing the liposomal antiretroviralliponucleotide drugs.

Antiviral Activity as Determined by HIV P24 Assay

Antiviral activity was assayed after 3 days by the inhibition of theproduction of HIV p24 (gag) antigen in the cell free culture medium ofthe infected cells exposed to different concentrations of drug; p24antigen was measured by ELISA (Abbott Laboratories, Chicago, Ill.)according to the manufacturer's instructions. The data are the averageof two determinations and are expressed as percentage of a controlincubated in the absence of drugs.

B. Experiment H533-1: FIG. 1

Liposomes containing 10 mole percent of eitherdimyristoylphosphatidylazidothymidine (LN1),dimyristoylphosphatidyldideoxythymidine (LN2) or azidothymidinediphosphate dimylristoylglycerol (LN4) in the indicated concentrationswere tested for their ability to inhibit HIV replication in CEM (wildtype) cells in vitro. All three of these antiretroviral liponucleotidesinhibited HIV p24 production; the amounts of drug required to reducevirus production by 50% (E.D. 50) were as follows:

Phosphatidylazidothymidine (LN1) 2 uM Phosphatidyldideoxythymidine (LN2)30 uM  AZT diphosphate dimyristoylglycerol (LN4) 8 uM

This demonstrates that the lipid derivatives of antiretroviralnucleotides can enter CEM cells and be converted to active nucleoside aspredicted. The control liposomes (CONT) which did not contain anyantiretroviral nucleotide had no effect on p24 production by CEM cells.

C. Experiment H747-1a: FIG. 2

Dimyristoylphosphatidylazidothymidine in liposomes (LN1) was comparedwith free azidothymidine (N1). At low concentrations below 0.1 uM freeAZT was more effective than the liponucleotide. At concentrationsranging from 2 to 170 uM the phosphatidylAZT liposomes were moreeffective than the free AZT. Control liposomes (CONT) containing onlyinactive lipids as noted in methods were ineffective in reducing p24.

D. Experiment H747-1b: FIG. 3

Dideoxythymidine (N2) is a weak inhibitor of HIV p24 production.Surprisingly, phosphatidyldideoxythymidine (LN2) is somewhat moreeffective than the free nucleoside. As can be seen in the chart,slightly more free ddT is required to reduce p24 production than withphosphatidyldideoxythymidine. Control liposomes (CONT) at a matchedtotal phospholipid concentration are without effect.

E. Experiment H637-1b: FIG. 4

In this experiment, CEM cells were replaced with mutant cells (providedby Dr. Dennis Carson, Scripps Clinic, San Diego, Calif.) which lack thethymidine kinase enzyme (CEM tk-). These cells are unable tophosphorylate thymidine derivatives and AZT is therefore inactive sinceit cannot be converted to the active triphosphate derivative which isneeded to inhibit HIV p24 replication. As shown in the chart, AZT (N1)is completely without effect on p24 production over a wide range ofconcentrations (0.2 to 100 uM). However, both phosphatidylAZT (LN1) andphosphatidylddT (LN2) were capable of reducing p24 production, provingthat these compounds are metabolized in the cell to thenucleoside-monophosphate which can be further activated to thetriphosphate by other cellular enzymes. This data provides proof of theprinciples outlined in the patent which predict direct metabolism to thenucleoside monophosphate.

F. Experiment H805-1: FIG. 5

In this experiment dimyristoylphosphatidyl-dideoxycytidine (LN3) anddimyristoyldideoxythymidine (LN2) were compared with the effects of freeAZT (N2) and dideoxycytidine (N3) in CEM (wild type) cells in vitro.PhosphatidylddC was the most potent liponucleotide (ED₅₀ 1.1 uM) andphosphatidylddT was less active as noted before (ED₅₀ 20 uM). Freeliposomes without added antiretroviral nucleotide (CONT) were inactive.

G. Experiment I276:

In this experiment, antiviral protection provided by preincubation withdimyristoylphosphatidylazidothymidine (LN1) in liposomes prepared asnoted above was compared with that of free azidothymidine (N1). CEM(wild type) cells were preincubated for 3 days under standard conditionsin RPMI media containing 7.14 μM of either free AZT (N1) orphosphatidylAZT (LN1). The cells were then washed twice with PBS, andfresh RPMI media added. Each group of cells was then divided into threebatches. One batch was immediately infected with HIV, as noted above;after washing away unattached HIV, the sample was allowed to incubate inmedia alone for 3 days. Two other batches were allowed to incubate inmedia alone for either 24 or 48 hours to allow any intracellularantiviral agent present to become depleted. Then they were infected withHIV, the cells washed free of virus, and fresh RPMI media added. After 3days of further incubation, the supernates of all batches were testedfor the presence of p24 protein.

Control Cells: CEM cells were subjected to HIV infection withoutpreincubation; drug was added following HIV infection as indicated, andthe cells were incubated for 3 days.

Preincubated Cells: CEM cells were preincubated for 3 days with mediacontaining AZT (N1) or phosphatidyl AZT (LN1); after 3 days the cellswere washed, subjected to HIV infection followed by addition of mediawithout drugs. After a further incubation for 3 days, p24 was measured.

Results

p24: ng/ml after 3 days CEM Controls: No Preincubation incubation HIVinfection only 204; 207 HIV + 7.14 μM Azidothymidine (N1)  64; 69 HIV +7.14 μM PhosphatidylAZT (LN1)  16; 16

Pre-Infection p24: ng/ml Interval after 3 days CEM Preincubated Cellswithout Drug incubation 7.14 μM Azidothymidine (N1) 24 h 404; 433 48 h271; 245 7.14 μM PhosphatidylAZT(LN1) 24 h  6; 7 48 h  4; 15

After a 3 day preincubation, followed by 48 hours of incubation innormal media after removal of the drugs, phosphatidylAZT providedcomplete protection from HIV replication as assessed by the reduced p24production. However, AZT preincubation failed to protect the cells fromHIV infection 24 and 48 hours after removal of the drug.

H. Experiment J45:

In this experiment the compound of Example 7(1-0-stearoylglycero-rac-3-phospho-5′-(3′-deoxy, 3′-azido) thymidine)was incorporated into liposomes containing 10 mole percent of theliponucleotide as indicated in Example 8. This material was diluted withRPMI medium to the desired concentration and added to HT4-6C cells (CD4+HeLa cells) obtained from Dr. Bruce Chesbro of the Rocky MountainNational Laboratories (Hamilton, Mont.) which had been infected withLAV-1 as noted earlier in this example. After a 3 day incubation at 37°C., the cells were washed with PBS, fixed and stained with crystalviolet and the plaques were counted. The results are shown below.

Liponucleotide Plaques, % of Untreated Concentration Average Control 10μM  1  2 3.16  7 13 1.0 16 29 0.316 32 58 0.100 39 71 0.0316 43 78 0 57— The data show that 1-0-stearoyl-rac-3-phospho-5′-(3′-deoxy,3′azido)thymidine is effective in inhibiting HIV plaque formation inHT4-6C cells infected with LAV-1. The concentration require to produce50% inhibition is about 0.35 micromolar.

EXAMPLE 11 Synthesis of Phosphatidylacyclovir and Efficacy in HerpesSimplex Virus-Infected WI-38 Cells

Dimyristoylphosphatidic acid (disodium salt) was obtained from AvantiPolar Lipids, Birmingham, Ala., and converted to the free acid (DMA-H)as described above in Example 1. Acycloguanosine (acyclovir, Zovirax®)was obtained from Sigma Chemical Co., St. Louis, Mo. and 73 mg (0.32mmol) was dried overnight over phosphorus pentoxide in a vacuum oven.250 mg of DMPA-H (0.42 mmol) was added to a 50 ml round bottom flask anddried overnight over phosphorus pentoxide in a vacuum oven. Under dryargon, 73 mg of acycloguanosine, 315 mg (1.04 mmol) oftriisopropylbenzenesulfonly chloride (Aldrich, Milwaukee, Wis.) and 2 mlof dry pyridine (Aldrich, Milwaukee, Wis.) were added to the roundbottom flask. The reaction mixture was stirred at room temperature for18 hours followed by the addition of 1 ml of distilled water.

The solvent was evaporated in vacuo to yield a yellow gum which wasredissolved in a small volume of chloroform/methanol (9/1) and appliedto a column of silica gel (45 gm: Kieselgel 60, EM Science, Cherry Hill,JN). The column was eluted with 8% methanol in chloroform (500 ml), 10%methanol in chloroform (250 ml) followed by 15% methanol in chloroform(1.5 L). After a 1.5 liter forerun rejected),dimyristoylphosphatidylacycloguanosine (pACV) was obtained. Threefractions were collected and analyzed: fraction 1 (200 ml, 130 mg pACV)continued pure pACV; fraction 2 (200 ml, 150 mg) and fraction 3 (200 ml,50 mg) contained pACV and small amounts of starting material asimpurities. Fraction 1 was concentrated in vacuo and to the residue wasadded 5 ml of cyclohexane; the solution was frozen and lyophilized todryness under phosphorus pentoxide to yield purephosphatidylacycloguanosine (80 mg, 0.1 mmol).

The purified compound gave a single spot with an Rf of 0.29 when appliedto K6G silica gel plates (Whatman International, Maidstone, England)developed with chloroform/methanol/water/ammonia (70/30/1 by volume).The UV absorption was maximal at 256 nm (extinction coefficient=8.4×10³in CHCl₃). The percentage phosphorus was 3.30% (theoretical 3.89%) andthe melting point was 245° C. On HPLC analysis,phosphatidylacycloguanosine gave a single peak with a retention time of11 minutes (Spheri-5; Brownlee Labs, Applied Biosystems, Santa Clara,Calif.) when eluted with a mobile phase of 1-propanol/0.25 mM potassiumphosphate/hexane/ethanol/acetic acid (245/179/31/50/0.5 by volume) at aflow rate of 0.5 ml/min.

Cell Cultures

Wi-38 cells were obtained from American Type Culture Collection(Rockville, Md. 20852) and grown in Dulbecco's minimum essential medium(DMEM) with 10% fetal calf serum (FCS). The cells were grown in 250 cmsquare bottles until reaching confluence.

Virus

Herpes simplex virus (HSV) type 1 (HSV-1) and type 2 (HSV-2) wereobtained from the American Type Culture Collection. Both virus stockswere prepared in Wi-38 cells; extensive cytopathic effects (CPD) wereobserved when the stock virus was harvested by a single freezing andthawing and the cell debris was clarified by low speed centrifugation(2000 rpm). Supernatant fluids containing the virus were aliquoted intosmall vials and stored at −80 C. Both HSV-1 and HSV-2 stocks weretitered in Wi-38 cells before use in the experiments.

Herpes Simples Virus Plague Reduction Assay

The plaque reduction assay was used to measure the antiviral effect ofphosphatidylacyclovir or free ACV. Wi-38 cells were trypsinized with0.25% trypsin for 5 min. The cells were harvested and centrifuged toremove residual trypsin and the cell pellet was resuspended in DMEM with10% FCS. The Wi-38 cells were plated in a 96 well plate (5×10cells/well) for one hour. The infected cells were then treated withphosphatidylacyclovir or ACV. The antiviral agents were prepared instock solutions which were then diluted two-fold with 2% FBS in DMEMcontaining 0.5% methylcellulose. 100 μl of each diluted antiviral agentwas added into each well of HSV infected cells.

The control and drug-treated cell cultures were incubated in a 37° C.incubator with 5% carbon dioxide for 24 hours. When HSV-infected cells(control without antiviral agent) showed readable number of plagues, theentire plate was fixed with methanol and stained with 1% crystal violetfor 10 min. The dye was rinsed off with tap water and the plate wasdried and plaques were counted. The antiviral effect of ACV orphosphatidylacyclovir was determined by measurement of plaque reductionas shown in the example below.

Results Effect if Acyclovir and Phosphatidylacyclovir on PlaqueFormation by HSV-1 IN WI-38 Cells

% no 1 2 mean Drug Acyclovir conc 10 uM 0 0 0  0 5 0 0 0  0 2.5 0 0 0  01.25 4 3 3.5 13 0.625 8 6 7 26 0.31 17  19  20 65 0.155 18  22  20 73 020;30 30;30 27.5 100  PhosphatidylACV 214 uM toxic toxic — — 107 0 0 0 0 54 0 0 0  0 27 2 3 2.5  9 13.4 4 6 5 18 6.7 6 9 7.5 27 3.3 10  12  1140 1.67 17  20  18.5 67 0.84 24  26  25 91 0 20;30 30;30 27.5 100 

The data shown above indicate that phosphatidylacyclovir is effective inHSV-1 infected Wi-38 cells. the concentration which produces 50%inhibition is 2 uM versus 0.4 uM for acyclovir. Similar results wereobtained with HSV-2 in infected Wi-38 cells.

EXAMPLE 12 Synthesis of 5′-palmitoyl(3′-deoxy-3′-azido)thymidine

0.5 grams of AZT (1.87 mmol) was dissolved in 10 ml of dry chloroformand 2 ml of dry pyridine. 0.78 grams (2.8 mmol) of palmitoyl chloride(Aldrich Chemicals, Milwaukee Wis.) dissolved in 5 ml of dry chloroformwas added slowly over a period of 20 minutes at 4° C. and the reactionmixture was allowed to warm to room temperature with stirring. After 20hours the reaction was stopped with the addition of 8 ml of distilledwater, and 38 ml of chloroform/methanol/0.5N HCl (1/2/0.8 by volume) wasadded. The phases were separated by the addition of 10 ml of chloroformand 8 ml of 0.5N HCl. The organic phase containing the required compoundwas further washed with 0.5N sodium bicarbonate. The lower chloroformphase was dried over anhydrous sodium sulfate and evaporated undervacuum. The compound was crystallized from chloroform/acetone at −20° C.Further purification was obtained by silicic acid column chromatography,and 145 mg of pure 5′-palmitoyl(3′-azido, 3′-deoxy)thymidine wasobtained (yield 15.3%). Elemental analysis: Predicted C 61.59, H 8.5, N13.8 and 0 15.8; Found C 60.74, H 8.6, N 13.5 and 0 17.9. Rf on silicagel G thin layer chromatography plates: 0.92(chloroform/methanol/ammonia/water, 70/30/1/1); 0.83(hexane/ethylether/acetic acid, 80/20/1) and 0.86 (chloroform/acetone,94/6), m.p. 77-80° C. ^(UV)max 265.

Efficacy of PalmitoylAZT in HIV-Infected HT4-6C Cells

PalmitoylAZT was incorporated into liposomes as noted in Example 8 andincubated with LAV-1 infected HT4-6C cells as noted in Examples 10 and11. 0.8 uM palmitoylAZT inhibited plaque formation by 25% (134 plaquesversus 176 in the untreated control).

It should be apparent from the foregoing that other nucleoside analoguesand phospholipid derivatives thereof can be substituted in the Examplesto obtain similar results. AZT-monophosphate or other antiviralnucleoside phosphate may also be contained in the aqueous compartmentsof the liposome. The molar percentage of the lipid antiviral nucleosidemay vary from 0.1 to 100% of the total lipid mixture. Furthermore,mixtures of antiviral nucleoside lipids may be used in constructing theliposomes for therapy of viral diseases. It should be further emphasizedthat the present invention is not limited to the use of any particularantiviral nucleoside analogue; rather, the beneficial results of thepresent invention flow from the formation of liposomes from the lipidderivatives of these materials. Thus, regardless of whether an antiviralnucleoside is presently known, or whether it becomes known in thefuture, the methods of forming the presently-contemplated lipidderivatives therefrom are based on established chemical techniques, aswill be apparent to those of skill in the art, and their incorporationinto liposomes is broadly enabled by the preceding disclosure. It shouldbe emphasized again that the present syntheses are broadly applicable toformation of compounds from essentially all nucleoside analogues for usein the practice of the present invention.

Accordingly, the invention may be embodied in other specific formswithout departing from it spirit or essential characteristics. Thedescribed embodiments are to be considered in all respects only asillustrative and not restrictive, and the scope of the invention is,therefore, indicated by the appended claims rather than by the foregoingdescription. All modifications which come within the meaning and rangeof the lawful equivalency of the claims are to be embraced with theirscope.

References

1. Richman, D. D., Kornbluth, R. S. and Carson, D. A. (1987) J. Exp.Med., 166: 1144-1149.

2. Fischl, M. S., Richman, D. D., Grieco, M. H., et al., (1987) New Eng.J. Med., 317: 185-191.

3. Richman, D. D., Fischl, M. A., Grieco, M. H., et al., (1987) New Eng.J. Med., 317: 192-197.

4. Bangham, A. D., Standish, M. M. and Watkins, J. C. (1965) J. Mol.Biol., 23: 238-252.

5. Black, C. D. V., Watson, G. J. and Ward, R. J. (1977) Trans. Roy.Soc. Trop. Med. Hyg., 71: 550-552.

6. Alving, C. R., Steck, E. A., Chapman, W. L., Waits, V. B., Hendricks,L. D. Swartz, G. M. and Hanson, W. L. (1978) Proc. Natl. Acad. Sci. USA75: 2959-2963.

7. Lopez-Berestein, G. (1986) Ann. Int. Med., 103: 694-699.

8. Herman, E. H., Rahman, A., Ferrans, V. J. Vick, J. A. and Shein, P.S. (1983) Cancer Res., 43: 5427-5432.

9. Ostro, M. (1987) Sci. Am. 256: 103-111.

10. Salahuddin, S. Z., Rose, R. M., Groopman, J. E., Markham, P. D. andGallo, R. C. (1985) Blood, 68: 281-284.

11. Koenig, S., Gendelman, H. E., Orenstein, J. M. Dalcanto, M. C.Pezeshkpur, G. H., Yungbluth, M. Janotta, F., Aksmit, A., Martin, M. A.and Fauci, A. S. (1986) Science, 233: 1089-1093.

12. Post, G., Kirsch, R. and Koestler, T. (1984) in Liposome Technology,Vol. III, G. Gregoriadis, Ed., CRC Press, Boca Raton, p. 1-28.

13. Scherphof, G. (1986) in Lipids and Biomernbranes, Past, Present andFuture, op den Kamp, J., Roelofsen, B. and Wirtz, K. W. A., Eds.,Elsevier North Holland, Amsterdam, p. 113-136.

14. Norley, S. G., Huang, L. and Rouse, B. T. (1987) J. Immunol., 136:681-685.

15. Kond, M., Alving, C. R., Rill, W. L., Swartz, G. M. and Cannonico,P. P. G. (1985) Antimicrob. Agents Chemother. 27: 903-907.

16. Matsushita, T., Ryu, E. K., Hong, C. I. and MacCoss, M. (1981)Cancer Res., 41: 2707-2713.

17. Ho, D. W. H. and Neil, B. L. (1977) Cancer Res., 37: 1640-1643.

18. Huang, A., Huang, L. and Kennel, S. J. (1980) J. Biol. Chem. 255:8015-8018.

19. Leserman, L. D., Barbet, J. and Kourilsky, F. (1980) Nature, 288:602-602.

20. Toorchen, D. and Topal, M. D. (1983) Carcinogenesis, 4: 1591-1597.

21. Agranoff, B. W. and Suomi, W. D. (1963) Biochem. Prep., 10: 46-51.

22. Prottey, C. and Hawthorne, J. N. (1967) Biochem. J., 105: 379-392.

23. Poorthuis, B. J. H. M. and Hostetler, K. Y. (1976) Biochim. Biophys.Acta, 431: 408-415.

24. ter Scheggett, J., van den Bosch, H., van Baak, M. A., Hostetler, K.Y. and Borst, P. (1971) Biochim. Biophys. Acta, 239: 234-242.

25. Rittenhouse, H. G., Seguin, E. B., Fisher, S. K. and Agranoff, B. W.(1981) J. Neurochem., 36: 991-999.

26. Olson, F., Hunt, C. A. Szoka, F. C., Vail, W. J. andPapahadjopoulos, D. (1979) Biochim, Biophys. Acta, 557: 9-23.

27. Szoka, F., and Papahadjopoulos, D. (1978) Proc. Nat. Acad. Sci. 75:4194-4198.

28. Mayhew, E., Lazo, R., Vail, W. J., King, J., Green, A. M. (1984)775: 169-175.

29. Kim, S., Turker, M., Chi, E., et al., Biochim. Biophys. Act, 728:339:348.

30. Mayer, L. D., Hope, M. J. and Cullis, P. R. (1986) Biochim. Biophys.Acta, 858: 161-168.

31. Fukanga, M., Miller, M. M., Hostetler, K. Y. and Deftos, L. J.(1984) Endocrinol. 115: 757-761.

32. Brown, D. A., Malkin T. and Maliphant, G. K. (1955) J. Chem. Soc.(London) pp. 1584-1588.

33. Heath, T. D., Lopez, N. G., Piper, J. R., Montgomery, J. A., Stern,W. H. and Papahadjopoulos, D. (1986) Biochim. Biophys. Acta, 862: 72-80.

34. Bligh, E. and Dyer, W. (1959) Canad. J. Biochem. Physiol.37:911-917.

35. Rosenthal, A. F. and Geyer, R. P.(1960) J. Biol. Chem. 235:2202.

What is claimed is:
 1. A method for inhibiting a viral polymerase in avirally infected mammalian cell without killing the cell, said methodcomprising administering to a mammal in need thereof an effectiveantiviral amount of a liponucleotide compound comprising: an antiviralnucleoside analogue having an ability to selectively inhibit said viralpolymerase enzyme without killing the cell; and a lipid moiety selectedfrom the group consisting of glycerolipids having the structure

 wherein said R₁ and R₂ independently have from 0 to 6 sites ofunsaturation, and have the structureCH₃—(CH₂)_(a)—(CH═CH—CH₂)_(b)—(CH₂)_(c)—Y wherein the sum of a and c isfrom 1 to 23; and b is 0 to 6; and wherein Y is selected from the groupconsisting of —CH₂—O, —CH═CH—O—, —CH₂—S—, and —CH═CH—S—; wherein theantiviral nucleoside analogue is covalently bound to the lipid moietythrough a mono-phosphate linkage to the 5′ carbon of the pentose residueof the nucleoside analogue corresponding to the 5′ carbon of naturallyoccurring nucleosides.
 2. The method of claim 1, wherein said nucleosideanalogue is selected from the group consisting of 2′,3′-dideoxycytidine;2′,3′-dideoxyguanosine; 2′,3′-dideoxyadenosine; 2′,3′-dideoxyinosine;2,6-diaminopurine 2′,3′-dideoxyriboside; 2′,3′-didehydrothymidine;2′,3′-didehydrocytidine carbocyclic; 2′,3′-didehydroguanosine;3′-azido-3′-deoxythymidine; 3′-azido-3′-deoxyguanosine;2,6-diaminopurine-3′-azido-2′,3′-dideoxyriboside;3′-fluoro-3′-deoxythymidine; 3′-fluoro-2′,3′-dideoxyguanosine;2′,3′-dideoxy-2′-fluoro-ara-adenosine;2,6-diaminopurine-3′-fluoro-2′,3′-dideoxyriboside;9-(4-hydroxy-1′,2′-butadienyl) adenine; 3-(4-hydroxy-1′,2′-butadienyl)cytosine; 9-(2-phosphonylmethoxyethyl) adenine;3-phosphonomethoxyethyl-2,6-diaminopurine; ganciclovir;3′-azido-2′,3′-dideoxy-5-chlorouridine (AzddClU);3′-azido-2′,3′-dideoxy-5-methylcytosine (AzddMeC);3′-azido-2′,3′-dideoxy-5-methylcytosine-N4OH (AzddMeC N4OH);3′-azido-2′,3′-dideoxy-5-methylcytosine-N4Me (AzddMeC N4Me);3′-azido-2′,3′-dideoxy-5-ethyluridine (AzddEtU);3′-azido-2′,3′-dideoxyuridine (AzddU); 3′-azido-2′,3′-dideoxycytosine(AzddC); 3′-azido-2′,3′-dideoxy-5-fluorocytosine (AzddFC);3′-azido-2′,3′-dideoxy-5-bromouridine (AzddBrU);3′-azido-2′,3′-dideoxy-5-fluorouridine (AzddTU);3′-fluoro-2′,3′-dideoxy-5-chlorouridine (FddClU);3′-fluoro-2′,3′-dideoxyuridine (3′FddU);3′-fluoro-2′,3′-dideoxythymidine (3′FddT);3′-fluoro-2′,3′-dideoxy-5-bromouridine (3′FddBrU);3′-fluoro-2′,3′-dideoxy-5-ethyluridine (3′FddEtU);2′,3′-dideoxy-2′,3′-didehydrothymidine (D4T);2′,3′-dideoxy-2′,3′-didehydrocytidine (D4C);2′,3′-dideoxy-2′,3′-didehydro-5-methylcytidine (D4MeC);2′,3′-dideoxy-2′,3′-didehydroadenosine (D4A);5-fluoro-2′,3′-dideoxycytidine (5FddC);2,6-diaminopurine-2′,3′-dideoxyriboside (ddDAPR);5-methyl-2′,3′-dideoxyadenosine (ddMeA);3′-azido-2′,3′-dideoxy-diaminopurine (N₃ddDAPR);3′-azido-2′,3′-dideoxyguanosine (3N₃ddG);3′-fluoro-2′,3′-dideoxy-diaminopurine (3FddDAPR);3′-fluoro-2′,3′-dideoxyguanosine (3FddG);3′-fluoro-2′,3′-dideoxy-arabinofuranosyl-adenine (3Fddara-A);3′-fluoro-2′,3′-dideoxyadenosine (3FddA); and2′,3′-dideoxy-3′thiacytidine (3TC).
 3. The method of claim 1 whereinsaid lipid moiety is selected from the group consisting of1,2-O-alkylglycerols and 1,2-S-alkylglycerols.
 4. The method of claim 1wherein said lipid moiety is selected from the group consisting of1-S-alkyl,2-O-alkylglycerols and 1-O-alkyl,2-S-alkylglycerols.
 5. Themethod of claim 1 wherein said inhibition of said viral polymerase bysaid liponucleotide is greater than an inhibition of said viralpolymerase by a corresponding free nucleoside.
 6. The method of claim 1wherein said liponucleotide comprises 1-S-dodecyl,2-O-decylglycero-phospho-3′-azido-3′deoxythymidine.
 7. The method ofclaim 1 wherein the antiviral nucleotide is1-S-dodecyl,2-O-decylglycero-phospho-2′,3′-dideoxycytosine.
 8. Themethod of claim 1 wherein the antiviral nucleotide is1-S-dodecyl,2-O-decylglycero-phospho-2′,3′-didehydro,2′,3′-dideoxythymidine.
 9. The method of claim 1 wherein the antiviralnucleotide is 1-S-dodecyl,2-O-decylglycero-phospho-2′,3′-dideoxyinosine.10. The method of claim 1 wherein the antiviral nucleotide is1-S-dodecyl,2-O-decylglycero-phospho-9-[(1,3-dihydroxy-2-propoxy)methyl]guanine.11. The method of claim 1 wherein the antiviral nucleotide is1-S-dodecyl,2-O-decylglycero-phospho-2′,3′-dideoxy-3′-thiacytidine.